Exon skipping oligomer conjugates for muscular dystrophy

ABSTRACT

Antisense oligomer conjugates complementary to a selected target site in the human dystrophin gene to induce exon 53 skipping are described.

FIELD OF THE DISCLOSURE

The present disclosure relates to novel antisense oligomer conjugatessuitable for exon 53 skipping in the human dystrophin gene andpharmaceutical compositions thereof. The disclosure also providesmethods for inducing exon 53 skipping using the novel antisense oligomerconjugates, methods for producing dystrophin in a subject having amutation of the dystrophin gene that is amenable to exon 53 skipping,and methods for treating a subject having a mutation of the dystrophingene that is amenable to exon 53 skipping.

BACKGROUND OF THE DISCLOSURE

Antisense technologies are being developed using a range of chemistriesto affect gene expression at a variety of different levels(transcription, splicing, stability, translation). Much of that researchhas focused on the use of antisense compounds to correct or compensatefor abnormal or disease-associated genes in a wide range of indications.Antisense molecules are able to inhibit gene expression withspecificity, and because of this, many research efforts concerningoligomers as modulators of gene expression have focused on inhibitingthe expression of targeted genes or the function of cis-acting elements.The antisense oligomers are typically directed against RNA, either thesense strand (e.g., mRNA), or minus-strand in the case of some viral RNAtargets. To achieve a desired effect of specific gene down-regulation,the oligomers generally either promote the decay of the targeted mRNA,block translation of the mRNA or block the function of cis-acting RNAelements, thereby effectively preventing either de novo synthesis of thetarget protein or replication of the viral RNA.

However, such techniques are not useful where the object is toup-regulate production of the native protein or compensate for mutationsthat induce premature termination of translation, such as nonsense orframe-shifting mutations. In these cases, the defective gene transcriptshould not be subjected to targeted degradation or steric inhibition, sothe antisense oligomer chemistry should not promote target mRNA decay orblock translation.

In a variety of genetic diseases, the effects of mutations on theeventual expression of a gene can be modulated through a process oftargeted exon skipping during the splicing process. The splicing processis directed by complex multi-component machinery that brings adjacentexon-intron junctions in pre-mRNA into close proximity and performscleavage of phosphodiester bonds at the ends of the introns with theirsubsequent reformation between exons that are to be spliced together.This complex and highly precise process is mediated by sequence motifsin the pre-mRNA that are relatively short, semi-conserved RNA segmentsto which various nuclear splicing factors that are then involved in thesplicing reactions bind. By changing the way the splicing machineryreads or recognizes the motifs involved in pre-mRNA processing, it ispossible to create differentially spliced mRNA molecules. It has nowbeen recognized that the majority of human genes are alternativelyspliced during normal gene expression, although the mechanisms involvedhave not been identified. Bennett et al. (U.S. Pat. No. 6,210,892)describe antisense modulation of wild-type cellular mRNA processingusing antisense oligomer analogs that do not induce RNAse H-mediatedcleavage of the target RNA. This finds utility in being able to generatealternatively spliced mRNAs that lack specific exons (see, e.g., asdescribed by Sazani, Kole, et al. 2007 for the generation of soluble TNFsuperfamily receptors that lack exons encoding membrane spanningdomains).

In cases where a normally functional protein is prematurely terminatedbecause of mutations therein, a means for restoring some functionalprotein production through antisense technology has been shown to bepossible through intervention during the splicing processes, and that ifexons associated with disease-causing mutations can be specificallydeleted from some genes, a shortened protein product can sometimes beproduced that has similar biological properties of the native protein orhas sufficient biological activity to ameliorate the disease caused bymutations associated with the exon (see e.g., Sierakowska, Sambade etal. 1996; Wilton, Lloyd et al. 1999; van Deutekom, Bremmer-Bout et al.2001; Lu, Mann et al. 2003; Aartsma-Rus, Janson et al. 2004). Kole etal. (U.S. Pat. Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593)disclose methods of combating aberrant splicing using modified antisenseoligomer analogs that do not promote decay of the targeted pre-mRNA.Bennett et al. (U.S. Pat. No. 6,210,892) describe antisense modulationof wild-type cellular mRNA processing also using antisense oligomeranalogs that do not induce RNAse H-mediated cleavage of the target RNA.

The process of targeted exon skipping is likely to be particularlyuseful in long genes where there are many exons and introns, where thereis redundancy in the genetic constitution of the exons or where aprotein is able to function without one or more particular exons.Efforts to redirect gene processing for the treatment of geneticdiseases associated with truncations caused by mutations in variousgenes have focused on the use of antisense oligomers that either: (1)fully or partially overlap with the elements involved in the splicingprocess; or (2) bind to the pre-mRNA at a position sufficiently close tothe element to disrupt the binding and function of the splicing factorsthat would normally mediate a particular splicing reaction which occursat that element.

Duchenne muscular dystrophy (DMD) is caused by a defect in theexpression of the protein dystrophin. The gene encoding the proteincontains 79 exons spread out over more than 2 million nucleotides ofDNA. Any exonic mutation that changes the reading frame of the exon, orintroduces a stop codon, or is characterized by removal of an entire outof frame exon or exons, or duplications of one or more exons, has thepotential to disrupt production of functional dystrophin, resulting inDMD.

A less severe form of muscular dystrophy, Becker muscular dystrophy(BMD) has been found to arise where a mutation, typically a deletion ofone or more exons, results in a correct reading frame along the entiredystrophin transcript, such that translation of mRNA into protein is notprematurely terminated. If the joining of the upstream and downstreamexons in the processing of a mutated dystrophin pre-mRNA maintains thecorrect reading frame of the gene, the result is an mRNA coding for aprotein with a short internal deletion that retains some activity,resulting in a Becker phenotype.

For many years it has been known that deletions of an exon or exonswhich do not alter the reading frame of a dystrophin protein would giverise to a BMD phenotype, whereas an exon deletion that causes aframe-shift will give rise to DMD (Monaco, Bertelson et al. 1988). Ingeneral, dystrophin mutations including point mutations and exondeletions that change the reading frame and thus interrupt properprotein translation result in DMD. It should also be noted that some BMDand DMD patients have exon deletions covering multiple exons.

Modulation of mutant dystrophin pre-mRNA splicing with antisenseoligoribonucleotides has been reported both in vitro and in vivo (seee.g., Matsuo, Masumura et al. 1991; Takeshima, Nishio et al. 1995;Pramono, Takeshima et al. 1996; Dunckley, Eperon et al. 1997; Dunckley,Manoharan et al. 1998; Wilton, Lloyd et al. 1999; Mann, Honeyman et al.2002; Errington, Mann et al. 2003).

Antisense oligomers have been specifically designed to target specificregions of the pre-mRNA, typically exons to induce the skipping of amutation of the DMD gene thereby restoring these out-of-frame mutationsin-frame to enable the production of internally shortened, yetfunctional dystrophin protein. Such antisense oligomers have been knownto target completely within the exon (so called exon internal sequences)or at a splice donor or splice acceptor junction that crosses from theexon into a portion of the intron.

The discovery and development of such antisense oligomers for DMD hasbeen an area of prior research. These developments include those from:(1) the University of Western Australia and Sarepta Therapeutics(assignee of this application): WO 2006/000057; WO 2010/048586; WO2011/057350; WO 2014/100714; WO 2014/153240; WO 2014/153220; (2)Academisch Ziekenhuis Leiden/Prosensa Technologies (now BioMarinPharmaceutical): WO 02/24906; WO 2004/083432; WO 2004/083446; WO2006/112705; WO 2007/133105; WO 2009/139630; WO 2009/054725; WO2010/050801; WO 2010/050802; WO 2010/123369; WO 2013/112053; WO2014/007620; (3) Carolinas Medical Center: WO 2012/109296; (4) RoyalHolloway: patents and applications claiming the benefit of, andincluding, US Ser. Nos. 61/096,073 and 61/164,978; such as U.S. Pat. No.8,084,601 and US 2017-0204413 (4) JCR Pharmaceuticals and Matsuo: U.S.Pat. No. 6,653,466; patents and applications claiming the benefit of,and including, JP 2000-125448, such as U.S. Pat. No. 6,653,467; patentsand applications claiming the benefit of, and including, JP 2000-256547,such as U.S. Pat. No. 6,727,355; WO 2004/048570; (5) Nippon Shinyaku: WO2012/029986; WO 2013/100190; WO 2015/137409; WO 2015/194520; and (6)Association Institut de Myologie/Universite Pierre et MarieCurie/Universitat Bern/Centre national de la RechercheScientifique/Synthena AG: WO 2010/115993; WO 2013/053928.

The discovery and development of antisense oligomers conjugated tocell-penetrating peptides for DMD has also been an area of research (seePCT Publication No. WO 2010/048586; Wu, B. et al., The American Journalof Pathology, Vol. 181 (2): 392-400, 2012; Wu, R. et al., Nucleic AcidsResearch, Vol. 35 (15): 5182-5191, 2007; Mulders, S. et al., 19^(th)International Congress of the World Muscle Society, Poster PresentationBerlin, October 2014; Bestas, B. et al., The Journal of ClinicalInvestigation, doi: 10.1172/JCI76175, 2014; Jearawiriyapaisarn, N. etal., Molecular Therapy, Vol. 16(9): 1624-1629, 2008; Jearawiriyapaisarn,N. et al., Cardiovascular Research, Vol. 85: 444-453, 2010; Moulton, H.M. et al., Biochemical Society Transactions, Vol. 35 (4): 826-828, 2007;Yin, H. et al., Molecular Therapy, Vol. 19 (7): 1295-1303, 2011; Abes,R. et al., J. Pept. Sci., Vol. 14: 455-460, 2008; Lebleu, B. et al.,Advanced Drug Delivery Reviews, Vol. 60: 517-529, 2008; McClorey, G. etal., Gene Therapy, Vol. 13: 1373-1381, 2006; Alter, J. et al., NatureMedicine, Vol. 12 (2): 175-177, 2006; and Youngblood, D. et al.,American Chemical Society, Bioconjugate Chem., 2007, 18 (1), pp 50-60).

Cell-penetrating peptides (CPP), for example, an arginine-rich peptidetransport moiety, may be effective to enhance penetration of, forexample, an antisense oligomer conjugated to the CPP, into a cell.

Despite these efforts, there remains a need for improved antisenseoligomers that target exon 53 and corresponding pharmaceuticalcompositions that are potentially useful for therapeutic methods forproducing dystrophin and treating DMD.

SUMMARY OF THE DISCLOSURE

The antisense oligomer conjugates provided herein include an antisenseoligomer moiety conjugated to a CPP. In one aspect, the disclosureprovides antisense oligomer conjugates comprising:

an antisense oligomer of 25 subunits in length capable of binding aselected target to induce exon skipping in the human dystrophin gene,wherein the antisense oligomer comprises a sequence of bases that iscomplementary to an exon 53 target region of the dystrophin pre-mRNAdesignated as an annealing site; and

a cell-penetrating peptide (CPP) conjugated to the antisense oligomer bya linker moiety.

In some embodiments, the annealing site is H53A(+36+60).

In some embodiments, the bases of the antisense oligomer are linked tomorpholino ring structures, wherein the morpholino ring structures arejoined by phosphorous-containing intersubunit linkages joining amorpholino nitrogen of one ring structure to a 5′ exocyclic carbon of anadjacent ring structure. In certain embodiments, the cell-penetratingpeptide is six arginine units (“R₆”) and the linker moiety is a glycine.In some embodiments, the antisense oligomer comprises a sequence ofbases designated as SEQ ID NO: 1.

In various aspects, the disclosure provides antisense oligomerconjugates which may be according to Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:each Nu is a nucleobase which taken together form a targeting sequence;and T is a moiety selected from:

R¹ is C₁-C₆ alkyl;

wherein the targeting sequence is complementary to an exon 53 annealingsite in the dystrophin pre-mRNA designated as H53A(+36+60).

In another aspect, the disclosure provides antisense oligomer conjugatesof Formula (IV):

or a pharmaceutically acceptable salt thereof.

In another aspect, the disclosure provides antisense oligomer conjugatesof Formula (IVA):

In another aspect, the disclosure provides pharmaceutical compositionsthat include the antisense oligomer conjugates of the disclosure, and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutically acceptable carrier is a saline solution that includes aphosphate buffer.

In another aspect, the disclosure provides a method for treatingDuchenne muscular dystrophy (DMD) in a subject in need thereof whereinthe subject has a mutation of the dystrophin gene that is amenable toexon 53 skipping, the method comprising administering to the subject anantisense oligomer conjugate of the disclosure. The disclosure alsoaddresses the use of antisense oligomer conjugates of the disclosure,for the manufacture of a medicament for treatment of Duchenne musculardystrophy (DMD) in a subject in need thereof wherein the subject has amutation of the dystrophin gene that is amenable to exon 53 skipping.

In another aspect, the disclosure provides a method of restoring an mRNAreading frame to induce dystrophin production in a subject having amutation of the dystrophin gene that is amenable to exon 53 skipping,the method comprising administering to the subject an antisense oligomerconjugate of the disclosure. In another aspect, the disclosure providesa method of excluding exon 53 from dystrophin pre-mRNA during mRNAprocessing in a subject having a mutation of the dystrophin gene that isamenable to exon 53 skipping, the method comprising administering to thesubject an antisense oligomer conjugate of the disclosure. In anotheraspect, the disclosure provides a method of binding exon 53 ofdystrophin pre-mRNA in a subject having a mutation of the dystrophingene that is amenable to exon 53 skipping, the method comprisingadministering to the subject an antisense oligomer conjugate of thedisclosure.

In another aspect, the disclosure provides an antisense oligomerconjugate of the disclosure herein for use in therapy. In certainembodiments, the disclosure provides an antisense oligomer conjugate ofthe disclosure for use in the treatment of Duchenne muscular dystrophy.In certain embodiments, the disclosure provides an antisense oligomerconjugate of the disclosure for use in the manufacture of a medicamentfor use in therapy. In certain embodiments, the disclosure provides anantisense oligomer conjugate of the disclosure for use in themanufacture of a medicament for the treatment of Duchenne musculardystrophy.

In another aspect, the disclosure also provides kits for treatingDuchenne muscular dystrophy (DMD) in a subject in need thereof whereinthe subject has a mutation of the dystrophin gene that is amenable toexon 53 skipping, which kits comprise at least an antisense oligomerconjugate of the present disclosure, packaged in a suitable containerand instructions for its use.

These and other objects and features will be more fully understood whenthe following detailed description of the disclosure is read inconjunction with the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a section of normal dystrophin pre-mRNA and mature mRNA.

FIG. 2 depicts a section of abnormal dystrophin pre-mRNA (example ofDMD) and resulting nonfunctional, unstable dystrophin.

FIG. 3 depicts eteplirsen, designed to skip exon 51, restoration of“In-frame” reading of pre-mRNA.

FIG. 4 provides a bar graph of the percentage of exon 53 skipping inhealthy human myoblasts by PMO #1 and PPMO #1 at various concentrations96 hours after treatment, as measured by RT-PCR. Error bars representmean±SD.

FIGS. 5A-5D provide representative images of Western Blot analysismeasuring dystrophin protein in the quadriceps of mdx mice treated withPMO (PMO4225) or PPMO (PPMO4225) for different time points [7 days (5A),30 days (5B), 60 days (5C), and 90 days (5D)].

FIG. 6A provides a line graph depicting the percentage of wild-typedystrophin induced by PMO (PMO4225) or PPMO (PPMO4225) in the quadricepsof mdx mice over 90 days post-injection, as determined by Western Blotanalysis.

FIG. 6B provides a line graph depicting the percentage of exon 23skipping induced by PMO (PMO4225) or PPMO (PPMO4225) in the quadricepsof mdx mice over 90 days post-injection, as determined by RT-PCR.

FIGS. 7A-7D provide representative images of Western Blot analysismeasuring dystrophin protein in the diaphragm of mdx mice treated withPMO (PMO4225) or PPMO (PPMO4225) for different time points [7 days (7A),30 days (7B), 60 days (7C) and 90 days (7D)].

FIG. 8A provides a line graph depicting the percentage of wild-typedystrophin induced by PMO (PMO4225) or PPMO (PPMO4225) in the diaphragmof mdx mice over 90 days post-injection, as determined by Western Blotanalysis.

FIG. 8B provides a line graph depicting the percentage of exon 23skipping induced by PMO (PMO4225) or PPMO (PPMO4225) in the diaphragm ofmdx mice over 90 days post-injection, as determined by RT-PCR.

FIG. 9A-9D provide representative images of Western Blot analysismeasuring dystrophin protein in the heart of mdx mice treated with PMO(PMO4225) or PPMO (PPMO4225) for different time points [7 days (9A), 30days (9B), 60 days (9C) and 90 days (9D)].

FIG. 10A provides a line graph depicting the percentage of wild-typedystrophin induced by PMO (PMO4225) or PPMO (PPMO4225) in the heart ofmdx mice over 90 days post-injection, as determined by Western Blotanalysis.

FIG. 10B provides a line graph depicting the percentage of exon 23skipping induced by PMO (PMO4225) or PPMO (PPMO4225) in the heart of mdxmice over 90 days post-injection, as determined by RT-PCR.

FIG. 11 provides immunohistochemistry analysis showing dystrophin in mdxmouse left quadriceps induced by PMO (PMO4225) or PPMO (PPMO4225).

FIG. 12A-B provide representative images of Western Blot analysismeasuring dystrophin protein in the heart of mdx mice treated with PMO(PMO4225) or PPMO (PPMO4225) for different doses: 40 mg/kg, 80 mg/kg,and 120 mg/kg.

FIG. 13 provides a bar graph depicting the percentage of wild-typedystrophin induced by PMO (PMO4225) or PPMO (PPMO4225) in the heart ofmdx mice as determined by Western Blot analysis 30 days post-injectionat different doses: 40 mg/kg, 80 mg/kg, and 120 mg/kg.

FIG. 14A-B provide representative images of Western Blot analysismeasuring dystrophin protein in the diaphragm of mdx mice treated withPMO (PMO4225) or PPMO (PPMO4225) for different doses 40 mg/kg, 80 mg/kg,and 120 mg/kg.

FIG. 15 provides a bar graph depicting the percentage of wild-typedystrophin induced by PMO (PMO4225) or PPMO (PPMO4225) in the diaphragmof mdx mice as determined by Western Blot analysis 30 dayspost-injection at different doses: 40 mg/kg, 80 mg/kg, and 120 mg/kg.

FIG. 16A-B provide representative images of Western Blot analysismeasuring dystrophin protein in the quadriceps of mdx mice treated withPMO (PMO4225) or PPMO (PPMO4225) at different doses: 40 mg/kg, 80 mg/kg,and 120 mg/kg.

FIG. 17 provides a bar graph depicting the percentage of wild-typedystrophin induced by PMO (PMO4225) or PPMO (PPMO4225) in the quadricepsof mdx mice as determined by Western Blot analysis 30 dayspost-injection at different doses: 40 mg/kg, 80 mg/kg, and 120 mg/kg.

FIG. 18 shows the coupling cycles performed by PMO Synthesis Method B.

FIG. 19 provides immunohistochemistry analysis showing dystrophin andlaminin in mdx mouse diaphragm and heart induced by PPMO (PPMO4225)compared to saline in mdx mice and wild type mice.

FIG. 20 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the quadriceps as determined by RT-PCR.

FIG. 21 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the diaphragm as determined by RT-PCR.

FIG. 22 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the heart as determined by RT-PCR.

FIG. 23 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the biceps as determined by RT-PCR.

FIG. 24 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the deltoid as determined by RT-PCR.

FIG. 25 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the aorta as determined by RT-PCR.

FIG. 26 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the duodenum as determined by RT-PCR.

FIG. 27 provides a line graph showing percent exon 53 skipping innon-human primates treated with PMO #1 or PPMO #1 weekly for four weeksat various doses. Percent exon 53 skipping was measured from musclesamples of the colon as determined by RT-PCR.

FIG. 28 provides a bar graph of the percentage of exon 53 skipping inhealthy human myotubes by PMO #1 and PPMO #1 at various concentrations96 hours after treatment, as measured by RT-PCR. Error bars representmean±SD.

DETAILED DESCRIPTION OF THE DISCLOSURE

Embodiments of the present disclosure relate generally to improvedantisense oligomer conjugates, and methods of use thereof, which arespecifically designed to induce exon skipping in the human dystrophingene. Dystrophin plays a vital role in muscle function, and variousmuscle-related diseases are characterized by mutated forms of this gene.Hence, in certain embodiments, the improved antisense oligomerconjugates described herein induce exon skipping in mutated forms of thehuman dystrophin gene, such as the mutated dystrophin genes found inDuchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

Due to aberrant mRNA splicing events caused by mutations, these mutatedhuman dystrophin genes either express defective dystrophin protein orexpress no measurable dystrophin at all, a condition that leads tovarious forms of muscular dystrophy. To remedy this condition, theantisense oligomer conjugates of the present disclosure hybridize toselected regions of a pre-processed mRNA of a mutated human dystrophingene, induce exon skipping and differential splicing in that otherwiseaberrantly spliced dystrophin mRNA, and thereby allow muscle cells toproduce an mRNA transcript that encodes a functional dystrophin protein.In certain embodiments, the resulting dystrophin protein is notnecessarily the “wild-type” form of dystrophin, but is rather atruncated, yet functional, form of dystrophin.

By increasing the levels of functional dystrophin protein in musclecells, these and related embodiments are useful in the prophylaxis andtreatment of muscular dystrophy, especially those forms of musculardystrophy, such as DMD and BMD, that are characterized by the expressionof defective dystrophin proteins due to aberrant mRNA splicing. Thespecific antisense oligomer conjugates described herein further provideimproved dystrophin-exon-specific targeting over other oligomers, andthereby offer significant and practical advantages over alternatemethods of treating relevant forms of muscular dystrophy.

Thus, the disclosure relates to antisense oligomer conjugatescomprising: an antisense oligomer of 25 subunits in length capable ofbinding a selected target to induce exon skipping in the humandystrophin gene, wherein the antisense oligomer comprises a sequence ofbases that is complementary to an exon 53 target region of thedystrophin pre-mRNA designated as an annealing site; and acell-penetrating peptide (CPP) conjugated to the antisense oligomer by alinker moiety.

In some embodiments, the annealing site is H53A(+36+60).

In some embodiments, the bases of the antisense oligomer are linked tomorpholino ring structures, wherein the morpholino ring structures arejoined by phosphorous-containing intersubunit linkages joining amorpholino nitrogen of one ring structure to a 5′ exocyclic carbon of anadjacent ring structure. In certain embodiments, the cell-penetratingpeptide is R6 and the linker moiety is a glycine. In some embodiments,the antisense oligomer comprises the sequence of bases designated as SEQID NO: 1, wherein each thymine base (T) is optionally a uracil base (U).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, preferred methods andmaterials are described. For the purposes of the present disclosure, thefollowing terms are defined below.

I. Definitions

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “alkyl,” as used herein, unless otherwise specified, refers toa saturated straight or branched hydrocarbon. In certain embodiments,the alkyl group is a primary, secondary, or tertiary hydrocarbon. Incertain embodiments, the alkyl group includes one to ten carbon atoms,i.e., C₁ to C₁₀ alkyl. In certain embodiments, the alkyl group includesone to six carbon atoms, i.e., C₁ to C₆ alkyl. In certain embodiments,the alkyl group is selected from the group consisting of methyl, CF₃,CCl₃, CFCl₂, CF₂Cl, ethyl, CH₂CF₃, CF₂CF₃, propyl, isopropyl, butyl,isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl,isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. Theterm includes both substituted and unsubstituted alkyl groups, includinghalogenated alkyl groups. In certain embodiments, the alkyl group is afluorinated alkyl group. Non-limiting examples of moieties with whichthe alkyl group can be substituted are selected from the groupconsisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

“Amenable to exon 53 skipping” as used herein with regard to a subjector patient is intended to include subjects and patients having one ormore mutations in the dystrophin gene which, absent the skipping of exon53 of the dystrophin pre-mRNA, causes the reading frame to beout-of-frame thereby disrupting translation of the pre-mRNA leading toan inability of the subject or patient to produce functional orsemi-functional dystrophin. Examples of mutations in the dystrophin genethat are amenable to exon 53 skipping include, e.g., deletion of: exons42 to 52, exons 45 to 52, exons 47 to 52, exons 48 to 52, exons 49 to52, exons 50 to 52, or exon 52. Determining whether a patient has amutation in the dystrophin gene that is amenable to exon skipping iswell within the purview of one of skill in the art (see, e.g.,Aartsma-Rus et al. (2009) Hum Mutat. 30:293-299; Gurvich et al., HumMutat. 2009; 30(4) 633-640; and Fletcher et al. (2010) Molecular Therapy18(6) 1218-1223.).

The term “oligomer” as used herein refers to a sequence of subunitsconnected by intersubunit linkages. In certain instances, the term“oligomer” is used in reference to an “antisense oligomer.” For“antisense oligomers,” each subunit consists of: (i) a ribose sugar or aderivative thereof; and (ii) a nucleobase bound thereto, such that theorder of the base-pairing moieties forms a base sequence that iscomplementary to a target sequence in a nucleic acid (typically an RNA)by Watson-Crick base pairing, to form a nucleic acid:oligomerheteroduplex within the target sequence with the proviso that either thesubunit, the intersubunit linkage, or both are not naturally occurring.In certain embodiments, the antisense oligomer is a PMO. In otherembodiments, the antisense oligomer is a 2′-O-methyl phosphorothioate.In other embodiments, the antisense oligomer of the disclosure is apeptide nucleic acid (PNA), a locked nucleic acid (LNA), or a bridgednucleic acid (BNA) such as 2′-0,4′-C-ethylene-bridged nucleic acid(ENA). Additional exemplary embodiments are described herein.

The terms “complementary” and “complementarity” refer to two or moreoligomers (i.e., each comprising a nucleobase sequence) that are relatedwith one another by Watson-Crick base-pairing rules. For example, thenucleobase sequence “T-G-A (5′-3′),” is complementary to the nucleobasesequence “A-C-T (3′ 4 5′).” Complementarity may be “partial,” in whichless than all of the nucleobases of a given nucleobase sequence arematched to the other nucleobase sequence according to base pairingrules. For example, in some embodiments, complementarity between a givennucleobase sequence and the other nucleobase sequence may be about 70%,about 75%, about 80%, about 85%, about 90% or about 95%. Or, there maybe “complete” or “perfect” (100%) complementarity between a givennucleobase sequence and the other nucleobase sequence to continue theexample. The degree of complementarity between nucleobase sequences hassignificant effects on the efficiency and strength of hybridizationbetween the sequences.

The terms “effective amount” and “therapeutically effective amount” areused interchangeably herein and refer to an amount of therapeuticcompound, such as an antisense oligomer, administered to a mammaliansubject, either as a single dose or as part of a series of doses, whichis effective to produce a desired therapeutic effect. For an antisenseoligomer, this effect is typically brought about by inhibitingtranslation or natural splice-processing of a selected target sequence,or producing a clinically meaningful amount of dystrophin (statisticalsignificance).

In some embodiments, an effective amount is at least 10 mg/kg, or atleast 20 mg/kg of a composition including an antisense oligomer for aperiod of time to treat the subject. In some embodiments, an effectiveamount is at least 20 mg/kg of a composition including an antisenseoligomer to increase the number of dystrophin-positive fibers in asubject to at least 20% of normal. In certain embodiments, an effectiveamount is 10 mg/kg, or at least at least 20 mg/kg of a compositionincluding an antisense oligomer to stabilize, maintain, or improvewalking distance from a 20% deficit, for example in a 6 MWT, in apatient, relative to a healthy peer. In various embodiments, aneffective amount is at least 10 mg/kg to about 30 mg/kg, at least 20mg/kg to about 30 mg/kg, about 25 mg/kg to about 30 mg/kg, or about 30mg/kg to about 50 mg/kg. In some embodiments, an effective amount isabout 10 mg/kg, about 20 mg/kg, about 30 mg/kg, or about 50 mg/kg. Inanother aspect, an effective amount is at least about 10 mg/kg, about 20mg/kg, about 25 mg/kg, about 30 mg/kg, or about 30 mg/kg to about 50mg/kg, for at least 24 weeks, at least 36 weeks, or at least 48 weeks,to thereby increase the number of dystrophin-positive fibers in asubject to at least 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95% of normal, and stabilize orimprove walking distance from a 20% deficit, for example in a 6 MWT, inthe patient relative to a healthy peer. In some embodiments, treatmentincreases the number of dystrophin-positive fibers to 20-60%, or 30-50%of normal in the patient.

By “enhance” or “enhancing,” or “increase” or “increasing,” or“stimulate” or “stimulating,” refers generally to the ability of one ormore antisense oligomer conjugates or pharmaceutical compositions toproduce or cause a greater physiological response (i.e., downstreameffects) in a cell or a subject, as compared to the response caused byeither no antisense oligomer conjugate or a control compound. A greaterphysiological response may include increased expression of a functionalform of a dystrophin protein, or increased dystrophin-related biologicalactivity in muscle tissue, among other responses apparent from theunderstanding in the art and the description herein. Increased musclefunction can also be measured, including increases or improvements inmuscle function by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Thepercentage of muscle fibers that express a functional dystrophin canalso be measured, including increased dystrophin expression in about 1%,2%, 5%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibers. Forinstance, it has been shown that around 40% of muscle functionimprovement can occur if 25-30% of fibers express dystrophin (see, e.g.,DelloRusso et al, Proc Natl Acad Sci USA 99: 12979-12984, 2002). An“increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000times, including all integers and decimal points in between and above1), e.g., 1.5, 1.6, 1.7, 1.8, etc.) the amount produced by no antisenseoligomer conjugate (the absence of an agent) or a control compound.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

A “functional” dystrophin protein refers generally to a dystrophinprotein having sufficient biological activity to reduce the progressivedegradation of muscle tissue that is otherwise characteristic ofmuscular dystrophy, typically as compared to the altered or “defective”form of dystrophin protein that is present in certain subjects with DMDor BMD. In certain embodiments, a functional dystrophin protein may haveabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (includingall integers in between) of the in vitro or in vivo biological activityof wild-type dystrophin, as measured according to routine techniques inthe art. As one example, dystrophin-related activity in muscle culturesin vitro can be measured according to myotube size, myofibrilorganization (or disorganization), contractile activity, and spontaneousclustering of acetylcholine receptors (see, e.g., Brown et al., Journalof Cell Science. 112:209-216, 1999). Animal models are also valuableresources for studying the pathogenesis of disease, and provide a meansto test dystrophin-related activity. Two of the most widely used animalmodels for DMD research are the mdx mouse and the golden retrievermuscular dystrophy (GRMD) dog, both of which are dystrophin negative(see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003). Theseand other animal models can be used to measure the functional activityof various dystrophin proteins. Included are truncated forms ofdystrophin, such as those forms that are produced following theadministration of certain of the exon-skipping antisense oligomerconjugates of the present disclosure.

The terms “mismatch” or “mismatches” refer to one or more nucleobases(whether contiguous or separate) in an oligomer nucleobase sequence thatare not matched to a target pre-mRNA according to base pairing rules.While perfect complementarity is often desired, some embodiments caninclude one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches withrespect to the target pre-mRNA. Variations at any location within theoligomer are included. In certain embodiments, antisense oligomerconjugates of the disclosure include variations in nucleobase sequencenear the termini variations in the interior, and if present aretypically within about 6, 5, 4, 3, 2, or 1 subunits of the 5′ and/or 3′terminus.

The terms “morpholino,” “morpholino oligomer,” and “PMO” refer to aphosphorodiamidate morpholino oligomer of the following generalstructure:

and as described in FIG. 2 of Summerton, J., et al., Antisense & NucleicAcid Drug Development, 7: 187-195 (1997). Morpholinos as describedherein include all stereoisomers and tautomers of the foregoing generalstructure. The synthesis, structures, and binding characteristics ofmorpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685;5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,521,063; 5,506,337;8,076,476; and 8,299,206; all of which are incorporated herein byreference.

In certain embodiments, a morpholino is conjugated at the 5′ or 3′ endof the oligomer with a “tail” moiety to increase its stability and/orsolubility. Exemplary tails include:

Of the above exemplary tail moieties, “TEG” or “EG3” refers to thefollowing tail moiety:

Of the above exemplary tail moieties, “GT” refers to the following tailmoiety:

As used herein, the terms “-G-R₆” and “-G-R₆-Ac” are usedinterchangeably and refer to a peptide moiety conjugated to an antisenseoligomer of the disclosure. In various embodiments, “G” represents aglycine residue conjugated to “R₆” by an amide bond, and each “R”represents an arginine residue conjugated together by amide bonds suchthat “R₆” means six (6) arginine residues conjugated together by amidebonds. The arginine residues can have any stereo configuration, forexample, the arginine residues can be L-arginine residues, D-arginineresidues, or a mixture of D- and L-arginine residues. In certainembodiments, “-G-R₆” or “-G-R₆-Ac” is conjugated to the morpholine ringnitrogen of the 3′ most morpholino subunit of a PMO antisense oligomerof the disclosure. In some embodiments, “-G-R₆” or “-G-R₆-Ac” isconjugated to the 3′ end of an antisense oligomer of the disclosure andis of the following formula:

The terms “nucleobase” (Nu), “base pairing moiety” or “base” are usedinterchangeably to refer to a purine or pyrimidine base found innaturally occurring, or “native” DNA or RNA (e.g., uracil, thymine,adenine, cytosine, and guanine), as well as analogs of these naturallyoccurring purines and pyrimidines. These analogs may confer improvedproperties, such as binding affinity, to the oligomer. Exemplary analogsinclude hypoxanthine (the base component of inosine); 2,6-diaminopurine;5-methyl cytosine; C5-propynyl-modified pyrimidines;10-(9-(aminoethoxy)phenoxazinyl) (G-clamp) and the like.

Further examples of base pairing moieties include, but are not limitedto, uracil, thymine, adenine, cytosine, guanine and hypoxanthine(inosine) having their respective amino groups protected by acylprotecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil,5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such aspseudoisocytosine and pseudouracil and other modified nucleobases suchas 8-substituted purines, xanthine, or hypoxanthine (the latter twobeing the natural degradation products). The modified nucleobasesdisclosed in: Chiu and Rana, R N A, 2003, 9, 1034-1048; Limbach et al.Nucleic Acids Research, 1994, 22, 2183-2196; and Revankar and Rao,Comprehensive Natural Products Chemistry, vol. 7, 313; are alsocontemplated, the contents of which are incorporated herein byreference.

Further examples of base pairing moieties include, but are not limitedto, expanded-size nucleobases in which one or more benzene rings hasbeen added. Nucleic acid base replacements described in: the GlenResearch catalog (www.glenresearch.com); Krueger A T et al., Acc. Chem.Res., 2007, 40, 141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943;Benner S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F.E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; and Hirao, I.,Curr. Opin. Chem. Biol., 2006, 10, 622-627; the contents of which areincorporated herein by reference, are contemplated as useful in theantisense oligomer conjugates described herein. Examples ofexpanded-size nucleobases include those shown below, as well astautomeric forms thereof.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

For clarity, structures of the disclosure including, for example,Formula (IV), are continuous from 5′ to 3′, and, for the convenience ofdepicting the entire structure in a compact form, various illustrationbreaks labeled “BREAK A,” “BREAK B,” and “BREAK C” have been included.As would be understood by the skilled artisan, for example, eachindication of “BREAK A” shows a continuation of the illustration of thestructure at these points. The skilled artisan understands that the sameis true for each instance of “BREAK B” and for “BREAK C” in thestructures above. None of the illustration breaks, however, are intendedto indicate, nor would the skilled artisan understand them to mean, anactual discontinuation of the structure above.

As used herein, a set of brackets used within a structural formulaindicate that the structural feature between the brackets is repeated.In some embodiments, the brackets used can be “[” and “],” and incertain embodiments, brackets used to indicate repeating structuralfeatures can be “(“and”).” In some embodiments, the number of repeatiterations of the structural feature between the brackets is the numberindicated outside the brackets such as 2, 3, 4, 5, 6, 7, and so forth.In various embodiments, the number of repeat iterations of thestructural feature between the brackets is indicated by a variableindicated outside the brackets such as “Z”.

As used herein, a straight bond or a squiggly bond drawn to a chiralcarbon or phosphorous atom within a structural formula indicates thatthe stereochemistry of the chiral carbon or phosphorous is undefined andis intended to include all forms of the chiral center. Examples of suchillustrations are depicted below.

The phrase “pharmaceutically acceptable” means the substance orcomposition must be compatible, chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the subject beingtreated therewith.

The phrase “pharmaceutically-acceptable carrier” as used herein means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material, or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are: sugars such as lactose, glucose, and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil;glycols such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; non-toxiccompatible lubricants such as sodium lauryl sulfate and magnesiumstearate; coloring agents; releasing agents; coating agents; sweeteningagents; flavoring agents; perfuming agents; preservatives; andantioxidants; according to the judgment of the formulator.

The term “restoration” with respect to dystrophin synthesis orproduction refers generally to the production of a dystrophin proteinincluding truncated forms of dystrophin in a patient with musculardystrophy following treatment with an antisense oligomer conjugatedescribed herein. In some embodiments, treatment results in an increasein novel dystrophin production in a patient by 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers inbetween). In some embodiments, treatment increases the number ofdystrophin-positive fibers to at least about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% to100% of normal in the subject. In other embodiments, treatment increasesthe number of dystrophin-positive fibers to about 20% to about 60%, orabout 30% to about 50%, of normal in the subject. The percent ofdystrophin-positive fibers in a patient following treatment can bedetermined by a muscle biopsy using known techniques. For example, amuscle biopsy may be taken from a suitable muscle, such as the bicepsbrachii muscle in a patient.

Analysis of the percentage of positive dystrophin fibers may beperformed pre treatment and/or post-treatment or at time pointsthroughout the course of treatment. In some embodiments, apost-treatment biopsy is taken from the contralateral muscle from thepre-treatment biopsy. Pre- and post-treatment dystrophin expressionanalysis may be performed using any suitable assay for dystrophin. Insome embodiments, immunohistochemical detection is performed on tissuesections from the muscle biopsy using an antibody that is a marker fordystrophin, such as a monoclonal or a polyclonal antibody. For example,the MANDYS 106 antibody can be used which is a highly sensitive markerfor dystrophin. Any suitable secondary antibody may be used.

In some embodiments, the percent dystrophin-positive fibers arecalculated by dividing the number of positive fibers by the total fiberscounted. Normal muscle samples have 100% dystrophin-positive fibers.Therefore, the percent dystrophin-positive fibers can be expressed as apercentage of normal. To control for the presence of trace levels ofdystrophin in the pretreatment muscle, as well as revertant fibers, abaseline can be set using sections of pre-treatment muscles from apatient when counting dystrophin-positive fibers in post-treatmentmuscles. This may be used as a threshold for countingdystrophin-positive fibers in sections of post-treatment muscle in thatpatient. In other embodiments, antibody-stained tissue sections can alsobe used for dystrophin quantification using Bioquant image analysissoftware (Bioquant Image Analysis Corporation, Nashville, Tenn.). Thetotal dystrophin fluorescence signal intensity can be reported as apercentage of normal. In addition, Western blot analysis with monoclonalor polyclonal anti-dystrophin antibodies can be used to determine thepercentage of dystrophin positive fibers. For example, theanti-dystrophin antibody NCL-Dys1 from Leica Biosystems may be used. Thepercentage of dystrophin-positive fibers can also be analyzed bydetermining the expression of the components of the sarcoglycan complex(β,γ) and/or neuronal NOS.

In some embodiments, treatment with an antisense oligomer conjugates ofthe disclosure slows or reduces the progressive respiratory muscledysfunction and/or failure in patients with DMD that would be expectedwithout treatment. In some embodiments, treatment with an antisenseoligomer conjugate of the disclosure may reduce or eliminate the needfor ventilation assistance that would be expected without treatment. Insome embodiments, measurements of respiratory function for tracking thecourse of the disease, as well as the evaluation of potentialtherapeutic interventions include maximum inspiratory pressure (MIP),maximum expiratory pressure (MEP), and forced vital capacity (FVC). MIPand MEP measure the level of pressure a person can generate duringinhalation and exhalation, respectively, and are sensitive measures ofrespiratory muscle strength. MIP is a measure of diaphragm muscleweakness.

In some embodiments, MEP may decline before changes in other pulmonaryfunction tests, including MIP and FVC. In certain embodiments, MEP maybe an early indicator of respiratory dysfunction. In certainembodiments, FVC may be used to measure the total volume of air expelledduring forced exhalation after maximum inspiration. In patients withDMD, FVC increases concomitantly with physical growth until the earlyteens. However, as growth slows or is stunted by disease progression,and muscle weakness progresses, the vital capacity enters a descendingphase and declines at an average rate of about 8 to 8.5 percent per yearafter 10 to 12 years of age. In certain embodiments, MIP percentpredicted (MIP adjusted for weight), MEP percent predicted (MEP adjustedfor age), and FVC percent predicted (FVC adjusted for age and height)are supportive analyses.

The terms “subject” and “patient” as used herein include any animal thatexhibits a symptom, or is at risk for exhibiting a symptom, which can betreated with an antisense oligomer conjugate of the disclosure, such asa subject (or patient) that has or is at risk for having DMD or BMD, orany of the symptoms associated with these conditions (e.g., muscle fiberloss). Suitable subjects (or patients) include laboratory animals (suchas mouse, rat, rabbit, or guinea pig), farm animals, and domesticanimals or pets (such as a cat or dog). Non-human primates and,preferably, human patients (or subjects), are included. Also includedare methods of producing dystrophin in a subject (or patient) having amutation of the dystrophin gene that is amenable to exon 53 skipping.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The phase “targeting sequence” refers to a sequence of nucleobases of anoligomer that is complementary to a sequence of nucleotides in a targetpre-mRNA. In some embodiments of the disclosure, the sequence ofnucleotides in the target pre-mRNA is an exon 53 annealing site in thedystrophin pre-mRNA designated as H53A(+36+60).

“Treatment” of a subject (e.g. a mammal, such as a human) or a cell isany type of intervention used in an attempt to alter the natural courseof the subject or cell. Treatment includes, but is not limited to,administration of an oligomer or a pharmaceutical composition thereof,and may be performed either prophylactically or subsequent to theinitiation of a pathologic event or contact with an etiologic agent.Treatment includes any desirable effect on the symptoms or pathology ofa disease or condition associated with the dystrophin protein, as incertain forms of muscular dystrophy, and may include, for example,minimal changes or improvements in one or more measurable markers of thedisease or condition being treated. Also included are “prophylactic”treatments, which can be directed to reducing the rate of progression ofthe disease or condition being treated, delaying the onset of thatdisease or condition, or reducing the severity of its onset. “Treatment”or “prophylaxis” does not necessarily indicate complete eradication,cure, or prevention of the disease or condition, or associated symptomsthereof.

In some embodiments, treatment with an antisense oligomer conjugate ofthe disclosure increases novel dystrophin production, delays diseaseprogression, slows or reduces the loss of ambulation, reduces muscleinflammation, reduces muscle damage, improves muscle function, reducesloss of pulmonary function, and/or enhances muscle regeneration thatwould be expected without treatment. In some embodiments, treatmentmaintains, delays, or slows disease progression. In some embodiments,treatment maintains ambulation or reduces the loss of ambulation. Insome embodiments, treatment maintains pulmonary function or reduces lossof pulmonary function. In some embodiments, treatment maintains orincreases a stable walking distance in a patient, as measured by, forexample, the 6 Minute Walk Test (6MWT). In some embodiments, treatmentmaintains or reduces the time to walk/run 10 meters (i.e., the 10 meterwalk/run test). In some embodiments, treatment maintains or reduces thetime to stand from supine (i.e, time to stand test). In someembodiments, treatment maintains or reduces the time to climb fourstandard stairs (i.e., the four-stair climb test). In some embodiments,treatment maintains or reduces muscle inflammation in the patient, asmeasured by, for example, MRI (e.g., MRI of the leg muscles). In someembodiments, MRI measures T2 and/or fat fraction to identify muscledegeneration. MM can identify changes in muscle structure andcomposition caused by inflammation, edema, muscle damage, and fatinfiltration.

In some embodiments, treatment with an antisense oligomer conjugate ofthe disclosure increases novel dystrophin production and slows orreduces the loss of ambulation that would be expected without treatment.For example, treatment may stabilize, maintain, improve or increasewalking ability (e.g., stabilization of ambulation) in the subject. Insome embodiments, treatment maintains or increases a stable walkingdistance in a patient, as measured by, for example, the 6 Minute WalkTest (6MWT), described by McDonald, et al. (Muscle Nerve, 2010;42:966-74, herein incorporated by reference). A change in the 6 MinuteWalk Distance (6MWD) may be expressed as an absolute value, a percentagechange or a change in the %-predicted value. In some embodiments,treatment maintains or improves a stable walking distance in a 6MWT froma 20% deficit in the subject relative to a healthy peer. The performanceof a DMD patient in the 6MWT relative to the typical performance of ahealthy peer can be determined by calculating a %-predicted value. Forexample, the %-predicted 6MWD may be calculated using the followingequation for males: 196.72+(39.81×age)−(1.36×age²)+(132.28×height inmeters). For females, the %-predicted 6MWD may be calculated using thefollowing equation: 188.61+(51.50×age)−(1.86×age²)+(86.10×height inmeters) (Henricson et al. PLoS Curr., 2012, version 2, hereinincorporated by reference). In some embodiments, treatment with anantisense oligomer increases the stable walking distance in the patientfrom baseline to greater than 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or50 meters (including all integers in between).

Loss of muscle function in patients with DMD may occur against thebackground of normal childhood growth and development. Indeed, youngerchildren with DMD may show an increase in distance walked during 6MWTover the course of about 1 year despite progressive muscular impairment.In some embodiments, the 6MWD from patients with DMD is compared totypically developing control subjects and to existing normative datafrom age and sex matched subjects. In some embodiments, normal growthand development can be accounted for using an age and height basedequation fitted to normative data. Such an equation can be used toconvert 6MWD to a percent-predicted (%-predicted) value in subjects withDMD. In certain embodiments, analysis of % predicted 6MWD datarepresents a method to account for normal growth and development, andmay show that gains in function at early ages (e.g., less than or equalto age 7) represent stable rather than improving abilities in patientswith DMD (Henricson et al. PLoS Curr., 2012, version 2, hereinincorporated by reference).

An antisense molecule nomenclature system was proposed and published todistinguish between the different antisense molecules (see Mann et al.,(2002) J Gen Med 4, 644-654). This nomenclature became especiallyrelevant when testing several slightly different antisense molecules,all directed at the same target region, as shown below: H #A/D(x:y).

The first letter designates the species (e.g. H: human, M: murine, C:canine). “#” designates target dystrophin exon number. “A/D” indicatesacceptor or donor splice site at the beginning and end of the exon,respectively. (x y) represents the annealing coordinates where “−” or“+” indicate intronic or exonic sequences respectively. For example,A(−6+18) would indicate the last 6 bases of the intron preceding thetarget exon and the first 18 bases of the target exon. The closestsplice site would be the acceptor so these coordinates would be precededwith an “A”. Describing annealing coordinates at the donor splice sitecould be D(+2-18) where the last 2 exonic bases and the first 18intronic bases correspond to the annealing site of the antisensemolecule. Entirely exonic annealing coordinates that would berepresented by A(+65+85), that is the site between the 65th and 85thnucleotide from the start of that exon.

II. Antisense Oligomers

A. Antisense Oligomer Conjugates Designed to Induce Exon 53 Skipping

In certain embodiments, antisense oligomer conjugates of the disclosureare complementary to an exon 53 target region of the dystrophin gene andinduce exon 53 skipping. In particular, the disclosure relates toantisense oligomer conjugates complementary to an exon 53 target regionof the dystrophin pre-mRNA designated as an annealing site. In someembodiments, the annealing site is H53A(+36+60).

Antisense oligomer conjugates of the disclosure target dystrophinpre-mRNA and induces skipping of exon 53, so it is excluded or skippedfrom the mature, spliced mRNA transcript. By skipping exon 53, thedisrupted reading frame is restored to an in-frame mutation. While DMDis comprised of various genetic subtypes, antisense oligomer conjugatesof the disclosure were specifically designed to skip exon 53 ofdystrophin pre-mRNA. DMD mutations amenable to skipping exon 53 comprisea subgroup of DMD patients (8%).

The nucleobase sequence of an antisense oligomer conjugate that inducesexon 53 skipping is designed to be complementary to a specific targetsequence within exon 53 of dystrophin pre-mRNA. In some embodiments, anantisense oligomer of the antisense oligomer conjugate is a PMO whereineach morpholino ring of the PMO is linked to a nucleobase including, forexample, nucleobases found in DNA (adenine, cytosine, guanine, andthymine).

B. Oligomer Chemistry Features

The antisense oligomer conjugates of the disclosure can employ a varietyof antisense oligomer chemistries. Examples of oligomer chemistriesinclude, without limitation, morpholino oligomers, phosphorothioatemodified oligomers, 2′ O-methyl modified oligomers, peptide nucleic acid(PNA), locked nucleic acid (LNA), phosphorothioate oligomers, 2′ 0-MOEmodified oligomers, 2′-fluoro-modified oligomer,2′O,4′C-ethylene-bridged nucleic acids (ENAs), tricyclo-DNAs,tricyclo-DNA phosphorothioate subunits, 2′-O-[2-(N-methylcarbamoyl)ethyl] modified oligomers, including combinations of any ofthe foregoing. Phosphorothioate and 2′-O-Me-modified chemistries can becombined to generate a 2′O-Me-phosphorothioate backbone. See, e.g., PCTPublication Nos. WO/2013/112053 and WO/2009/008725, which are herebyincorporated by reference in their entireties. Exemplary embodiments ofoligomer chemistries of the disclosure are further described below.

1. Peptide Nucleic Acids (PNAs)

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligomers obeying Watson-Crick base-pairing rules, andmimic DNA in terms of base pair recognition (Egholm, Buchardt et al.1993). The backbone of PNAs is formed by peptide bonds rather thanphosphodiester bonds, making them well-suited for antisense applications(see structure below). The backbone is uncharged, resulting in PNA/DNAor PNA/RNA duplexes that exhibit greater than normal thermal stability.PNAs are not recognized by nucleases or proteases. A non-limitingexample of a PNA is depicted below.

Despite a radical structural change to the natural structure, PNAs arecapable of sequence-specific binding in a helix form to DNA or RNA.Characteristics of PNAs include a high binding affinity to complementaryDNA or RNA, a destabilizing effect caused by single-base mismatch,resistance to nucleases and proteases, hybridization with DNA or RNAindependent of salt concentration and triplex formation with homopurineDNA. PANAGENE™ has developed its proprietary Bts PNA monomers (Bts;benzothiazole-2-sulfonyl group) and proprietary oligomerization process.The PNA oligomerization using Bts PNA monomers is composed of repetitivecycles of deprotection, coupling and capping. PNAs can be producedsynthetically using any technique known in the art. See, e.g., U.S. Pat.Nos. 6,969,766; 7,211,668; 7,022,851; 7,125,994; 7,145,006; and7,179,896. See also U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262for the preparation of PNAs. Further teaching of PNA compounds can befound in Nielsen et al., Science, 254:1497-1500, 1991. Each of theforegoing is incorporated by reference in its entirety.

2. Locked Nucleic Acids (LNAs)

Antisense oligomer conjugates may also contain “locked nucleic acid”subunits (LNAs). “LNAs” are a member of a class of modifications calledbridged nucleic acid (BNA). BNA is characterized by a covalent linkagethat locks the conformation of the ribose ring in a C30-endo (northern)sugar pucker. For LNA, the bridge is composed of a methylene between the2′-O and the 4′-C positions. LNA enhances backbone preorganization andbase stacking to increase hybridization and thermal stability.

The structures of LNAs can be found, for example, in Wengel, et al.,Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998)54:3607; Jesper Wengel, Accounts of Chem. Research (1999) 32:301; Obika,et al., Tetrahedron Letters (1997) 38:8735; Obika, et al., TetrahedronLetters (1998) 39:5401; and Obika, et al., Bioorganic MedicinalChemistry (2008) 16:9230, which are hereby incorporated by reference intheir entirety. A non-limiting example of an LNA is depicted below.

Antisense oligomer conjugates of the disclosure may incorporate one ormore LNAs; in some cases, the antisense oligomer conjugates may beentirely composed of LNAs. Methods for the synthesis of individual LNAnucleoside subunits and their incorporation into oligomers aredescribed, for example, in U.S. Pat. Nos. 7,572,582; 7,569,575;7,084,125; 7,060,809; 7,053,207; 7,034,133; 6,794,499; and 6,670,461;each of which is incorporated by reference in its entirety. Typicalintersubunit linkers include phosphodiester and phosphorothioatemoieties; alternatively, non-phosphorous containing linkers may beemployed. Further embodiments include an LNA containing antisenseoligomer conjugate where each LNA subunit is separated by a DNA subunit.Certain antisense oligomer conjugates are composed of alternating LNAand DNA subunits where the intersubunit linker is phosphorothioate.

2′O,4′C-ethylene-bridged nucleic acids (ENAs) are another member of theclass of BNAs. A non-limiting example is depicted below.

ENA oligomers and their preparation are described in Obika et al.,Tetrahedron Lett (1997) 38 (50): 8735, which is hereby incorporated byreference in its entirety. Antisense oligomer conjugates of thedisclosure may incorporate one or more ENA subunits.

3. Unlocked Nucleic Acid (UNA)

Antisense oligomer conjugates may also contain unlocked nucleic acid(UNA) subunits. UNAs and UNA oligomers are an analogue of RNA in whichthe C2′-C3′ bond of the subunit has been cleaved. Whereas LNA isconformationally restricted (relative to DNA and RNA), UNA is veryflexible. UNAs are disclosed, for example, in WO 2016/070166. Anon-limiting example of an UNA is depicted below.

Typical intersubunit linkers include phosphodiester and phosphorothioatemoieties; alternatively, non-phosphorous containing linkers may beemployed.

4. Phosphorothioates

“Phosphorothioates” (or S-oligos) are a variant of normal DNA in whichone of the nonbridging oxygens is replaced by a sulfur. A non-limitingexample of a phosphorothioate is depicted below.

The sulfurization of the internucleotide bond reduces the action ofendo- and exonucleases including 5′ to 3′ and 3′ to 5′ DNA POL 1exonuclease, nucleases S1 and P1, RNases, serum nucleases and snakevenom phosphodiesterase. Phosphorothioates are made by two principalroutes: by the action of a solution of elemental sulfur in carbondisulfide on a hydrogen phosphonate, or by the method of sulfurizingphosphite triesters with either tetraethylthiuram disulfide (TETD) or3H-1, 2-bensodithiol-3-one 1, 1-dioxide (BDTD) (see, e.g., Iyer et al.,J. Org. Chem. 55, 4693-4699, 1990, which is hereby incorporated byreference in its entirety). The latter methods avoid the problem ofelemental sulfur's insolubility in most organic solvents and thetoxicity of carbon disulfide. The TETD and BDTD methods also yieldhigher purity phosphorothioates.

5. Triclyclo-DNAs and Tricyclo-Phosphorothioate Subunits

Tricyclo-DNAs (tc-DNA) are a class of constrained DNA analogs in whicheach nucleotide is modified by the introduction of a cyclopropane ringto restrict conformational flexibility of the backbone and to optimizethe backbone geometry of the torsion angle γ. Homobasic adenine- andthymine-containing tc-DNAs form extraordinarily stable A-T base pairswith complementary RNAs. Tricyclo-DNAs and their synthesis are describedin International Patent Application Publication No. WO 2010/115993,which is hereby incorporated by reference in its entirety. Antisenseoligomer conjugates of the disclosure may incorporate one or moretricycle-DNA subunits; in some cases, the antisense oligomer conjugatesmay be entirely composed of tricycle-DNA subunits.

Tricyclo-phosphorothioate subunits are tricyclo-DNA subunits withphosphorothioate intersubunit linkages. Tricyclo-phosphorothioatesubunits and their synthesis are described in International PatentApplication Publication No. WO 2013/053928, which is hereby incorporatedby reference in its entirety. Antisense oligomer conjugates of thedisclosure may incorporate one or more tricycle-DNA subunits; in somecases, the antisense oligomer conjugates may be entirely composed oftricycle-DNA subunits. A non-limiting example of atricycle-DNA/tricycle-phophothioate subunit is depicted below.

6. 2′ 0-Methyl, 2′ O-MOE, and 2′-F Oligomers

“2′-O-Me oligomer” molecules carry a methyl group at the 2′-OH residueof the ribose molecule. 2′-O-Me-RNAs show the same (or similar) behavioras DNA, but are protected against nuclease degradation. 2′-O-Me-RNAs canalso be combined with phosphorothioate oligomers (PTOs) for furtherstabilization. 2′O-Me oligomers (phosphodiester or phosphothioate) canbe synthesized according to routine techniques in the art (see, e.g.,Yoo et al., Nucleic Acids Res. 32:2008-16, 2004, which is herebyincorporated by reference in its entirety). A non-limiting example of a2′ 0-Me oligomer is depicted below.

2′ O-Methoxyethyl Oligomers (2′-O MOE) carry a methoxyethyl group at the2′-OH residue of the ribose molecule and are discussed in Martin et al.,Helv. Chim. Acta, 78, 486-504, 1995, which is hereby incorporated byreference in its entirety. A non-limiting example of a 2′O MOE subunitis depicted below.

2′-Fluoro (2′-F) oligomers have a fluoro radical in at the 2′ positionin place of the 2′OH. A non-limiting example of a 2′-F oligomer isdepicted below.

2′-fluoro oligomers are further described in WO 2004/043977, which ishereby incorporated by reference in its entirety.

2′O-Methyl, 2′ O-MOE, and 2′-F oligomers may also comprise one or morephosphorothioate (PS) linkages as depicted below.

Additionally, 2′O-Methyl, 2′ O-MOE, and 2′-F oligomers may comprise PSintersubunit linkages throughout the oligomer, for example, as in the2′O-methyl PS oligomer drisapersen depicted below.

Alternatively, 2′ 0-Methyl, 2′ O-MOE, and/or 2′-F oligomers may comprisePS linkages at the ends of the oligomer, as depicted below.

where:

R is CH₂CH₂OCH₃ (methoxyethyl or MOE); and

x, y, and z denote the number of nucleotides contained within each ofthe designated 5′-wing, central gap, and 3′-wing regions, respectively.

Antisense oligomer conjugates of the disclosure may incorporate one ormore 2′ 0-Methyl, 2′ O-MOE, and 2′-F subunits and may utilize any of theintersubunit linkages described here. In some instances, an antisenseoligomer conjugate of the disclosure may be composed of entirely2′O-Methyl, 2′ O-MOE, or 2′-F subunits. One embodiment of an antisenseoligomer conjugates of the disclosure is composed entirely of 2′O-methylsubunits.

7. 2′-O-[2-(N-methylcarbamoyl)ethyl] Oligomers (MCEs)

MCEs are another example of 2′O modified ribonucleosides useful in theantisense oligomer conjugates of the disclosure. Here, the 2′OH isderivatized to a 2-(N-methylcarbamoyl)ethyl moiety to increase nucleaseresistance. A non-limiting example of an MCE oligomer is depicted below.

MCEs and their synthesis are described in Yamada et al., J. Org. Chem.(2011) 76(9):3042-53, which is hereby incorporated by reference in itsentirety. Antisense oligomer conjugates of the disclosure mayincorporate one or more MCE subunits.

8. Stereo Specific Oligomers

Stereo specific oligomers are those in which the stereo chemistry ofeach phosphorous-containing linkage is fixed by the method of synthesissuch that a substantially stereo-pure oligomer is produced. Anon-limiting example of a stereo specific oligomer is depicted below.

In the above example, each phosphorous of the oligomer has the samestereo configuration. Additional examples include the oligomersdescribed above. For example, LNAs, ENAs, Tricyclo-DNAs, MCEs, 2′O-Methyl, 2′O-MOE, 2′-F, and morpholino-based oligomers can be preparedwith stereo-specific phosphorous-containing internucleoside linkagessuch as, for example, phosphorothioate, phosphodiester, phosphoramidate,phosphorodiamidate, or other phosphorous-containing internucleosidelinkages. Stereo specific oligomers, methods of preparation, chiralcontrolled synthesis, chiral design, and chiral auxiliaries for use inpreparation of such oligomers are detailed, for example, inWO2017192664, WO2017192679, WO2017062862, WO2017015575, WO2017015555,WO2015107425, WO2015108048, WO2015108046, WO2015108047, WO2012039448,WO2010064146, WO2011034072, WO2014010250, WO2014012081, WO20130127858,and WO2011005761, each of which is hereby incorporated by reference inits entirety.

Stereo specific oligomers can have phosphorous-containinginternucleoside linkages in an R_(P) or S_(P) configuration. Chiralphosphorous-containing linkages in which the stereo configuration of thelinkages is controlled is referred to as “stereopure,” while chiralphosphorous-containing linkages in which the stereo configuration of thelinkages is uncontrolled is referred to as “stereorandom.” In certainembodiments, the oligomers of the disclosure comprise a plurality ofstereopure and stereorandom linkages, such that the resulting oligomerhas stereopure subunits at pre-specified positions of the oligomer. Anexample of the location of the stereopure subunits is provided ininternational patent application publication number WO 2017/062862 A2 inFIGS. 7A and 7B. In an embodiment, all the chiral phosphorous-containinglinkages in an oligomer are stereorandom. In an embodiment, all thechiral phosphorous-containing linkages in an oligomer are stereopure.

In an embodiment of an oligomer with n chiral phosphorous-containinglinkages (where n is an integer of 1 or greater), all n of the chiralphosphorous-containing linkages in the oligomer are stereorandom. In anembodiment of an oligomer with n chiral phosphorous-containing linkages(where n is an integer of 1 or greater), all n of the chiralphosphorous-containing linkages in the oligomer are stereopure. In anembodiment of an oligomer with n chiral phosphorous-containing linkages(where n is an integer of 1 or greater), at least 10% (to the nearestinteger) of the n phosphorous-containing linkages in the oligomer arestereopure. In an embodiment of an oligomer with n chiralphosphorous-containing linkages (where n is an integer of 1 or greater),at least 20% (to the nearest integer) of the n phosphorous-containinglinkages in the oligomer are stereopure. In an embodiment of an oligomerwith n chiral phosphorous-containing linkages (where n is an integer of1 or greater), at least 30% (to the nearest integer) of the nphosphorous-containing linkages in the oligomer are stereopure. In anembodiment of an oligomer with n chiral phosphorous-containing linkages(where n is an integer of 1 or greater), at least 40% (to the nearestinteger) of the n phosphorous-containing linkages in the oligomer arestereopure. In an embodiment of an oligomer with n chiralphosphorous-containing linkages (where n is an integer of 1 or greater),at least 50% (to the nearest integer) of the n phosphorous-containinglinkages in the oligomer are stereopure. In an embodiment of an oligomerwith n chiral phosphorous-containing linkages (where n is an integer of1 or greater), at least 60% (to the nearest integer) of the nphosphorous-containing linkages in the oligomer are stereopure. In anembodiment of an oligomer with n chiral phosphorous-containing linkages(where n is an integer of 1 or greater), at least 70% (to the nearestinteger) of the n phosphorous-containing linkages in the oligomer arestereopure. In an embodiment of an oligomer with n chiralphosphorous-containing linkages (where n is an integer of 1 or greater),at least 80% (to the nearest integer) of the n phosphorous-containinglinkages in the oligomer are stereopure. In an embodiment of an oligomerwith n chiral phosphorous-containing linkages (where n is an integer of1 or greater), at least 90% (to the nearest integer) of the nphosphorous-containing linkages in the oligomer are stereopure.

In an embodiment of an oligomer with n chiral phosphorous-containinglinkages (where n is an integer of 1 or greater), the oligomer containsat least 2 contiguous stereopure phosphorous-containing linkages of thesame stereo orientation (i.e. either S_(P) or R_(P)). In an embodimentof an oligomer with n chiral phosphorous-containing linkages (where n isan integer of 1 or greater), the oligomer contains at least 3 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 4 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 5 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 6 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 7 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 8 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 9 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 10 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 11 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 12 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 13 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 14 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 15 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 16 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 17 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 18 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 19 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)). In an embodiment of anoligomer with n chiral phosphorous-containing linkages (where n is aninteger of 1 or greater), the oligomer contains at least 20 contiguousstereopure phosphorous-containing linkages of the same stereoorientation (i.e. either S_(P) or R_(P)).

9. Morpholino Oligomers

Exemplary embodiments of the disclosure relate to phosphorodiamidatemorpholino oligomers of the following general structure:

and as described in FIG. 2 of Summerton, J., et al., Antisense & NucleicAcid Drug Development, 7: 187-195 (1997). Morpholinos as describedherein are intended to cover all stereoisomers and tautomers of theforegoing general structure. The synthesis, structures, and bindingcharacteristics of morpholino oligomers are detailed in U.S. Pat. Nos.5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,521,063;5,506,337; 8,076,476; and 8,299,206, all of which are incorporatedherein by reference.

In certain embodiments, a morpholino is conjugated at the 5′ or 3′ endof the oligomer with a “tail” moiety to increase its stability and/orsolubility. Exemplary tails include:

In various embodiments, an antisense oligomer conjugate of thedisclosure is according to Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

each Nu is a nucleobase which taken together form a targeting sequence;T is a moiety selected from:

R¹ is C₁-C₆ alkyl;

wherein the targeting sequence is complementary to an exon 53 annealingsite in the dystrophin pre-mRNA designated as H53A(+36+60).

In various embodiments, T is

In various embodiments, R¹ is methyl, CF₃, CCl₃, CFCl₂, CF₂Cl, ethyl,CH₂CF₃, CF₂CF₃, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl,pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl,2,2-dimethylbutyl, or 2,3-dimethylbutyl.

In some embodiments, an antisense oligomer conjugate of Formula (I) isan HCl (hydrochloric acid) salt thereof. In certain embodiments, the HClsalt is a 0.6HCl salt.

In some embodiments, each Nu is independently selected from cytosine(C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5 mC),uracil (U), and hypoxanthine (I).

In some embodiments, the targeting sequence is SEQ ID NO: 1(5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′), wherein each thymine (T) isoptionally uracil (U).

In various embodiments, T is

and the targeting sequence is SEQ ID NO: 1(5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′), wherein each thymine (T) isoptionally uracil (U).

In various embodiments, T is

and the targeting sequence is SEQ ID NO: 1(5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′).

In some embodiments, including, for example, some embodiments of Formula(I), an antisense oligomer conjugate of the disclosure is according toFormula (II):

or a pharmaceutically acceptable salt thereof, wherein:

each Nu is a nucleobase which taken together form a targeting sequencethat is complementary to an exon 53 annealing site in the dystrophinpre-mRNA designated as H53A(+36+60).

In some embodiments, each Nu is independently selected from cytosine(C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5 mC),uracil (U), and hypoxanthine (I).

In various embodiments, each Nu from 1 to 25 and 5′ to 3′ is (SEQ ID NO:1):

Position No. 5’ to 3’ Nu 1 G 2 X 3 X 4 G 5 C 6 C 7 X 8 C 9 C 10 G 11 G12 X 13 X 14 C 15 X 16 G 17 A 18 A 19 G 20 G 21 X 22 G 23 X 24 X 25 C

wherein A is

C is

G is

and X is

In certain embodiments, each X is independently

In Some embodiments, an antisense oligomer conjugate of Formula (II) isan HCl (hydrochloric acid) salt thereof. In certain embodiments, the HClsalt is a 0.6HCl salt.

In some embodiments, including, for example, some embodiments of Formula(II), an antisense oligomer conjugate of the disclosure is according toFormula (IIA):

wherein each Nu is a nucleobase which taken together form a targetingsequence that is complementary to an exon 53 annealing site in thedystrophin pre-mRNA designated as H53A(+36+60).

In some embodiments, each Nu is independently selected from cytosine(C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5 mC),uracil (U), and hypoxanthine (I).

In various embodiments, each Nu from 1 to 25 and 5′ to 3′ is (SEQ ID NO:1):

Position No. 5’ to 3’ Nu 1 G 2 X 3 X 4 G 5 C 6 C 7 X 8 C 9 C 10 G 11 G12 X 13 X 14 C 15 X 16 G 17 A 18 A 19 G 20 G 21 X 22 G 23 X 24 X 25 C

wherein A is

C is

G is

and X is

In certain embodiments, each X is

In some embodiments including, for example, embodiments of antisenseoligomer conjugates of Formula (II) and Formula (IIA), the targetingsequence is SEQ ID NO: 1 (5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′) wherein eachthymine (T) is optionally uracil (U). In various embodiments including,for example, embodiments of antisense oligomer conjugates of Formula(II) and Formula (IIA), the targeting sequence is SEQ ID NO: 1(5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′).

In some embodiments, including, for example, embodiments of antisenseoligomer conjugates of Formula (I), an antisense oligomer conjugate ofthe disclosure is according to Formula (III):

or a pharmaceutically acceptable salt thereof.

In some embodiments, an antisense oligomer conjugate of Formula (III) isan HCl (hydrochloric acid) salt thereof. In certain embodiments, the HClsalt is a 0.6HCl salt.

In some embodiments, including, for example, embodiments of antisenseoligomer conjugates of Formula (III), an antisense oligomer conjugate ofthe disclosure is according to Formula (IIIA):

In some embodiments of the disclosure, including some embodiments ofantisense oligomer conjugates of Formula (I) and embodiments ofantisense oligomer conjugates of Formula (III), the antisense oligomerconjugate is according to Formula (IV): or a pharmaceutically acceptablesalt thereof.

In some embodiments, an antisense oligomer conjugate of Formula (IV) isan HCl (hydrochloric acid) salt thereof. In certain embodiments, the HClsalt is a 0.6HCl salt.

In some embodiments, including, for example, embodiments of antisenseoligomer conjugates of Formula (IV), an antisense oligomer conjugate ofthe disclosure is according to Formula (IVA):

10. Nucleobase Modifications and Substitutions

In certain embodiments, antisense oligomer conjugates of the disclosureare composed of RNA nucleobases and DNA nucleobases (often referred toin the art simply as “base”). RNA bases are commonly known as adenine(A), uracil (U), cytosine (C) and guanine (G). DNA bases are commonlyknown as adenine (A), thymine (T), cytosine (C) and guanine (G). Invarious embodiments, antisense oligomer conjugates of the disclosure arecomposed of cytosine (C), guanine (G), thymine (T), adenine (A),5-methylcytosine (5 mC), uracil (U), and hypoxanthine (I).

In some embodiments, an antisense oligomer conjugate of the disclosurecomprises a sequence of nucleobases which taken together form atargeting sequence wherein the targeting sequence is complementary to anexon 53 annealing site in the dystrophin pre-mRNA designated asH53A(+36+60). In various embodiments, the sequence of nucleobasescomprises the sequence of SEQ ID NO: 1. In some embodiments, thesequence of nucleobases consists of the sequence of SEQ ID NO: 1. Invarious embodiments, the sequence of nucleobases comprises a sequencehaving deletion, substitution, insertion and/or addition of 1 to 5nucleobases in the sequence of SEQ ID NO:1. In various embodiments, thesequence of nucleobases consists of a sequence having deletion,substitution, insertion and/or addition of 1 to 5 nucleobases in thesequence of SEQ ID NO:1. In various embodiments, the targeting sequencehas a region complementary to at least one string of three or moreidentical contiguous nucleobases in annealing sight, wherein theannealing site comprises at least one additional nucleobase compared tothe region of the targeting sequence and the at least one additionalnucleobase has no complementary nucleobase in the region of thetargeting sequence, and wherein the targeting region complementary tothe at least one string of three or more identical contiguousnucleobases is internal to the targeting sequence, examples of which canbe found in U.S. Patent Ser. No. 62/573,985, which is incorporated hereentirely by reference.

In certain embodiments, one or more RNA bases or DNA bases in anoligomer may be modified or substituted with a base other than a RNAbase or DNA base. Oligomers containing a modified or substituted baseinclude oligomers in which one or more purine or pyrimidine bases mostcommonly found in nucleic acids are replaced with less common ornon-natural bases.

Purine bases comprise a pyrimidine ring fused to an imidazole ring, asdescribed by the following general formula.

Adenine and guanine are the two purine nucleobases most commonly foundin nucleic acids. Other naturally-occurring purines include, but notlimited to, N⁶-methyladenine, N²-methylguanine, hypoxanthine, and7-methylguanine.

Pyrimidine bases comprise a six-membered pyrimidine ring as described bythe following general formula.

Cytosine, uracil, and thymine are the pyrimidine bases most commonlyfound in nucleic acids. Other naturally-occurring pyrimidines include,but not limited to, 5-methylcytosine, 5-hydroxymethylcytosine,pseudouracil, and 4-thiouracil. In one embodiment, the oligomersdescribed herein contain thymine bases in place of uracil.

Other suitable bases include, but are not limited to: 2,6-diaminopurine,orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g. 2-thiouracil,2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidines(e.g. 5-halouracil, 5-propynyluracil, 5-propynylcytosine,5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine,5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine,7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine,8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine,or derivatives thereof; N²-cyclopentylguanine (cPent-G),N²-cyclopentyl-2-aminopurine (cPent-AP), and N²-propyl-2-aminopurine(Pr-AP), pseudouracil, or derivatives thereof; and degenerate oruniversal bases, like 2,6-difluorotoluene or absent bases like abasicsites (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose;or pyrrolidine derivatives in which the ring oxygen has been replacedwith nitrogen (azaribose)). Examples of derivatives of Super A, Super G,and Super T can be found in U.S. Pat. No. 6,683,173 (Epoch Biosciences),which is incorporated here entirely by reference. cPent-G, cPent-AP, andPr-AP were shown to reduce immunostimulatory effects when incorporatedin siRNA (Peacock H. et al. J. Am. Chem. Soc. 2011, 133, 9200).Pseudouracil is a naturally occurring isomerized version of uracil, witha C-glycoside rather than the regular N-glycoside as in uridine.Pseudouridine-containing synthetic mRNA may have an improved safetyprofile compared to uridine-containing mPvNA (WO 2009127230,incorporated here in its entirety by reference).

Certain nucleobases are particularly useful for increasing the bindingaffinity of the antisense oligomer conjugates of the disclosure. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and0-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-0-methoxyethyl sugar modifications. Additionalexemplary modified nucleobases include those wherein at least onehydrogen atom of the nucleobase is replaced with fluorine.

11. Pharmaceutically Acceptable Salts of Antisense Oligomer Conjugates

Certain embodiments of antisense oligomer conjugates described hereinmay contain a basic functional group, such as amino or alkylamino, andare, thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of antisense oligomer conjugates of thepresent disclosure. These salts can be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byseparately reacting a purified antisense oligomer conjugate of thedisclosure in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed during subsequent purification.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,lactobionate, and laurylsulphonate salts and the like. (See, e.g., Bergeet al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject antisense oligomerconjugates include the conventional nontoxic salts or quaternaryammonium salts of the antisense oligomer conjugates, e.g., fromnon-toxic organic or inorganic acids. For example, such conventionalnontoxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic,and the like.

In certain embodiments, the antisense oligomer conjugates of the presentdisclosure may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non toxic, inorganicand organic base addition salts of antisense oligomer conjugates of thepresent disclosure. These salts can likewise be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byseparately reacting the purified antisense oligomer conjugate in itsfree acid form with a suitable base, such as the hydroxide, carbonate,or bicarbonate of a pharmaceutically-acceptable metal cation, withammonia, or with a pharmaceutically-acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, e.g., Berge et al., supra).

III. Formulations and Modes of Administration

In certain embodiments, the present disclosure provides formulations orpharmaceutical compositions suitable for the therapeutic delivery ofantisense oligomer conjugates, as described herein. Hence, in certainembodiments, the present disclosure provides pharmaceutically acceptablecompositions that comprise a therapeutically-effective amount of one ormore of the antisense oligomer conjugates described herein, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. While it is possible for an antisenseoligomer conjugate of the present disclosure to be administered alone,it is preferable to administer the antisense oligomer conjugate as apharmaceutical formulation (composition). In an embodiment, theantisense oligomer conjugate of the formulation is according to Formula(III).

Methods for the delivery of nucleic acid molecules, which can beapplicable to the antisense oligomer conjugates of the presentdisclosure, are described, for example, in: Akhtar et al., 1992, TrendsCell Bio., 2:139; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995, CRC Press; and Sullivan et al., PCT WO94/02595. These and other protocols can be utilized for the delivery ofvirtually any nucleic acid molecule, including the antisense oligomerconjugates of the present disclosure.

The pharmaceutical compositions of the present disclosure may bespecially formulated for administration in solid or liquid form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets (targeted for buccal, sublingual, or systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous, or epidural injection as, for example, a sterile solutionor suspension, or sustained-release formulation; (3) topicalapplication, for example, as a cream, ointment, or a controlled-releasepatch or spray applied to the skin; (4) intravaginally or intrarectally,for example, as a pessary, cream, or foam; (5) sublingually; (6)ocularly; (7) transdermally; or (8) nasally.

Some examples of materials that can serve as pharmaceutically-acceptablecarriers include, without limitation: (1) sugars, such as lactose,glucose and sucrose; (2) starches, such as corn starch and potatostarch; (3) cellulose, and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such ascocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates, and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Additional non-limiting examples of agents suitable for formulation withthe antisense oligomer conjugates of the instant disclosure include: PEGconjugated nucleic acids; phospholipid conjugated nucleic acids; nucleicacids containing lipophilic moieties; phosphorothioates; P-glycoproteininhibitors (such as Pluronic P85) which can enhance entry of drugs intovarious tissues; biodegradable polymers, such as poly(D,L-lactide-coglycolide) microspheres for sustained release deliveryafter implantation (Emerich, D F et al., 1999, Cell Transplant, 8,47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, suchas those made of polybutylcyanoacrylate, which can deliver drugs acrossthe blood brain barrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

The disclosure also features the use of the composition comprisingsurface-modified liposomes containing poly(ethylene glycol) (“PEG”)lipids (PEG-modified, branched and unbranched or combinations thereof,or long-circulating liposomes or stealth liposomes). Oligomer conjugatesof the disclosure can also comprise covalently attached PEG molecules ofvarious molecular weights. These formulations offer a method forincreasing the accumulation of drugs in target tissues. This class ofdrug carriers resists opsonization and elimination by the mononuclearphagocytic system (MPS or RES), thereby enabling longer bloodcirculation times and enhanced tissue exposure for the encapsulated drug(Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem.Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown toaccumulate selectively in tumors, presumably by extravasation andcapture in the neovascularized target tissues (Lasic et al., Science1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238,86-90). The long-circulating liposomes enhance the pharmacokinetics andpharmacodynamics of DNA and RNA, particularly compared to conventionalcationic liposomes which are known to accumulate in tissues of the MPS(Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al.,International PCT Publication No. WO 96/10391; Ansell et al.,International PCT Publication No. WO 96/10390; Holland et al.,International PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

In a further embodiment, the present disclosure includes antisenseoligomer conjugate pharmaceutical compositions prepared for delivery asdescribed in U.S. Pat. Nos. 6,692,911; 7,163,695; and 7,070,807. In thisregard, in one embodiment, the present disclosure provides an antisenseoligomer conjugate of the present disclosure in a composition comprisingcopolymers of lysine and histidine (HK) (as described in U.S. Pat. Nos.7,163,695; 7,070,807; and 6,692,911) either alone or in combination withPEG (e.g., branched or unbranched PEG or a mixture of both), incombination with PEG and a targeting moiety, or any of the foregoing incombination with a crosslinking agent. In certain embodiments, thepresent disclosure provides antisense oligomer conjugates inpharmaceutical compositions comprising gluconic-acid-modifiedpolyhistidine or gluconylated-polyhistidine/transferrin-polylysine. Oneskilled in the art will also recognize that amino acids with propertiessimilar to His and Lys may be substituted within the composition.

Wetting agents, emulsifiers and lubricants (such as sodium laurylsulfate and magnesium stearate), coloring agents, release agents,coating agents, sweetening agents, flavoring agents, perfuming agents,preservatives, and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present disclosure include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient that canbe combined with a carrier material to produce a single dosage form willvary depending upon the subject being treated and the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the active ingredient which produces a therapeuticeffect. Generally this amount will range from about 0.1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

In certain embodiments, a formulation of the present disclosurecomprises an excipient selected from cyclodextrins, celluloses,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and an antisense oligomerconjugate of the present disclosure. In an embodiment, the antisenseoligomer conjugate of the formulation is according to Formula (III). Incertain embodiments, an aforementioned formulation renders orallybioavailable an antisense oligomer conjugate of the present disclosure.

Methods of preparing these formulations or pharmaceutical compositionsinclude the step of bringing into association an antisense oligomerconjugate of the present disclosure with the carrier and, optionally,one or more accessory ingredients. In general, the formulations areprepared by uniformly and intimately bringing into association anantisense oligomer conjugate of the present disclosure with liquidcarriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the disclosure suitable for oral administration may bein the form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of an antisense oligomerconjugate of the present disclosure as an active ingredient. Anantisense oligomer conjugate of the present disclosure may also beadministered as a bolus, electuary, or paste.

In solid dosage forms of the disclosure for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient may be mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid pharmaceutical compositions of a similar type may also beemployed as fillers in soft and hard-shelled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (e.g., gelatin or hydroxypropylmethyl cellulose), lubricant,inert diluent, preservative, disintegrant (for example, sodium starchglycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present disclosure, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid pharmaceutical compositions whichcan be dissolved in sterile water, or some other sterile injectablemedium immediately before use. These pharmaceutical compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms for oral administration of the antisense oligomerconjugates of the disclosure include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral pharmaceutical compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, coloring, perfuming and preservativeagents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more compounds ofthe disclosure with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations or dosage forms for the topical or transdermaladministration of an oligomer as provided herein include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active oligomer conjugates may be mixed under sterileconditions with a pharmaceutically-acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to an activecompound of this disclosure, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an antisense oligomerconjugate of the present disclosure, excipients such as lactose, talc,silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of an antisense oligomer conjugate of the present disclosure tothe body. Such dosage forms can be made by dissolving or dispersing theoligomer in the proper medium. Absorption enhancers can also be used toincrease the flux of the agent across the skin. The rate of such fluxcan be controlled by either providing a rate controlling membrane ordispersing the agent in a polymer matrix or gel, among other methodsknown in the art.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more oligomer conjugates of the disclosure incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the disclosure include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. In an embodiment, theantisense oligomer conjugate of the pharmaceutical composition isaccording to Formula (III).

These pharmaceutical compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms upon the subject oligomerconjugates may be ensured by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility, amongother methods known in the art. The rate of absorption of the drug thendepends upon its rate of dissolution which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

Injectable depot forms may be made by forming microencapsule matrices ofthe subject oligomer conjugates in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of oligomer topolymer, and the nature of the particular polymer employed, the rate ofoligomer release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations may also prepared by entrapping the drug inliposomes or microemulsions that are compatible with body tissues.

When the antisenseoligomer conjugates of the present disclosure areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99% (more preferably, 10 to 30%) of the antisense oligomerconjugate in combination with a pharmaceutically acceptable carrier.

The formulations or preparations of the present disclosure may be givenorally, parenterally, topically, or rectally. They are typically givenin forms suitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, or infusion; topically by lotion orointment; or rectally by suppositories.

Regardless of the route of administration selected, the antisenseoligomer conjugates of the present disclosure, which may be used in asuitable hydrated form, and/or the pharmaceutical compositions of thepresent disclosure, may be formulated into pharmaceutically-acceptabledosage forms by conventional methods known to those of skill in the art.Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this disclosure may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being unacceptably toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular antisense oligomer conjugate ofthe present disclosure employed, or the ester, salt or amide thereof,the route of administration, the time of administration, the rate ofexcretion or metabolism of the particular oligomer being employed, therate and extent of absorption, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular oligomer employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the antisense oligomer conjugates of the disclosureemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, a suitable daily dose of an antisense oligomer conjugate of thedisclosure will be that amount of the antisense oligomer conjugate whichis the lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described herein.Generally, oral, intravenous, intracerebroventricular and subcutaneousdoses of the antisense oligomer conjugates of this disclosure for apatient, when used for the indicated effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day.

In some embodiments, the antisense oligomer conjugates of the presentdisclosure are administered in doses generally from about 10-160 mg/kgor 20-160 mg/kg. In some cases, doses of greater than 160 mg/kg may benecessary. In some embodiments, doses for i.v. administration are fromabout 0.5 mg to 160 mg/kg. In some embodiments, the antisense oligomerconjugates are administered at doses of about 0.5 mg/kg, 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or10 mg/kg. In some embodiments, the antisense oligomer conjugates areadministered at doses of about 10 mg/kg, 11 mg/kg, 12 mg/kg, 15 mg/kg,18 mg/kg, 20 mg/kg, 21 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg 50mg/kg, 51 mg/kg, 52 mg/kg, 53 mg/kg, 54 mg/kg, 55 mg/kg, 56 mg/kg, 57mg/kg, 58 mg/kg, 59 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110 mg/kg,115 mg/kg, 120 mg/kg, 125 mg/kg, 130 mg/kg, 135 mg/kg, 140 mg/kg, 145mg/kg, 150 mg/kg, 155 mg/kg, 160 mg/kg, including all integers inbetween. In some embodiments, the oligomer is administered at 10 mg/kg.In some embodiments, the oligomer is administered at 20 mg/kg. In someembodiments, the oligomer is administered at 30 mg/kg. In someembodiments, the oligomer is administered at 40 mg/kg. In someembodiments, the oligomer is administered at 60 mg/kg. In someembodiments, the oligomer is administered at 80 mg/kg. In someembodiments, the oligomer is administered at 160 mg/kg. In someembodiments, the oligomer is administered at 50 mg/kg.

In some embodiments, the antisense oligomer conjugate of Formula (III)is administered in doses generally from about 10-160 mg/kg or 20-160mg/kg. In some embodiments, doses of the antisense oligomer conjugate ofFormula (III) for i.v. administration are from about 0.5 mg to 160mg/kg. In some embodiments, the antisense oligomer conjugate of Formula(III) is administered at doses of about 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10mg/kg. In some embodiments, the antisense oligomer conjugate of Formula(III) is administered at doses of about 10 mg/kg, 11 mg/kg, 12 mg/kg, 15mg/kg, 18 mg/kg, 20 mg/kg, 21 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49mg/kg 50 mg/kg, 51 mg/kg, 52 mg/kg, 53 mg/kg, 54 mg/kg, 55 mg/kg, 56mg/kg, 57 mg/kg, 58 mg/kg, 59 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110mg/kg, 115 mg/kg, 120 mg/kg, 125 mg/kg, 130 mg/kg, 135 mg/kg, 140 mg/kg,145 mg/kg, 150 mg/kg, 155 mg/kg, 160 mg/kg, including all integers inbetween. In some embodiments, the antisense oligomer conjugate ofFormula (III) is administered at 10 mg/kg. In some embodiments, theantisense oligomer conjugate of Formula (III) is administered at 20mg/kg. In some embodiments, the antisense oligomer conjugate of Formula(III) is administered at 30 mg/kg. In some embodiments, the antisenseoligomer conjugate of Formula (III) is administered at 40 mg/kg. In someembodiments, the antisense oligomer conjugate of Formula (III) isadministered at 60 mg/kg. In some embodiments, the antisense oligomerconjugate of Formula (III) is administered at 80 mg/kg. In someembodiments, the antisense oligomer conjugate of Formula (III) isadministered at 160 mg/kg. In some embodiments, the antisense oligomerconjugate of Formula (III) is administered at 50 mg/kg.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain situations, dosing is oneadministration per day. In certain embodiments, dosing is one or moreadministration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to maintain thedesired expression of a functional dystrophin protein. In certainembodiments, dosing is one or more administrations once every two weeks.In some embodiments, dosing is one administration once every two weeks.In various embodiments, dosing is one or more administrations everymonth. In certain embodiments, dosing is one administration every month.

In various embodiments, the antisense oligomer conjugates areadministered weekly at 10 mg/kg. In various embodiments, the antisenseoligomer conjugates are administered weekly at 20 mg/kg. In variousembodiments, the antisense oligomer conjugates are administered weeklyat 30 mg/kg. In various embodiments, the antisense oligomer conjugatesare administered weekly at 40 mg/kg. In some embodiments, the antisenseoligomer conjugates are administered weekly at 60 mg/kg. In someembodiments, the antisense oligomer conjugates are administered weeklyat 80 mg/kg. In some embodiments, the antisense oligomer conjugates areadministered weekly at 100 mg/kg. In some embodiments, the antisenseoligomer conjugates are administered weekly at 160 mg/kg. As usedherein, weekly is understood to have the art-accepted meaning of everyweek.

In various embodiments, the antisense oligomer conjugates areadministered biweekly at 10 mg/kg. In various embodiments, the antisenseoligomer conjugates are administered biweekly at 20 mg/kg. In variousembodiments, the antisense oligomer conjugates are administered biweeklyat 30 mg/kg. In various embodiments, the antisense oligomer conjugatesare administered biweekly at 40 mg/kg. In some embodiments, theantisense oligomer conjugates are administered biweekly at 60 mg/kg. Insome embodiments, the antisense oligomer conjugates are administeredbiweekly at 80 mg/kg. In some embodiments, the antisense oligomerconjugates are administered biweekly at 100 mg/kg. In some embodiments,the antisense oligomer conjugates are administered biweekly at 160mg/kg. As used herein, biweekly is understood to have the art-acceptedmeaning of every two weeks.

In various embodiments, the antisense oligomer conjugates areadministered every third week at 10 mg/kg. In various embodiments, theantisense oligomer conjugates are administered every third week at 20mg/kg. In various embodiments, the antisense oligomer conjugates areadministered every third week at 30 mg/kg. In various embodiments, theantisense oligomer conjugates are administered every third week at 40mg/kg. In some embodiments, the antisense oligomer conjugates areadministered every third week at 60 mg/kg. In some embodiments, theantisense oligomer conjugates are administered every third week at 80mg/kg. In some embodiments, the antisense oligomer conjugates areadministered every third week at 100 mg/kg. In some embodiments, theantisense oligomer conjugates are administered every third week at 160mg/kg. As used herein, every third week is understood to have theart-accepted meaning of once every three weeks.

In various embodiments, the antisense oligomer conjugates areadministered monthly at 10 mg/kg. In various embodiments, the antisenseoligomer conjugates are administered monthly at 20 mg/kg. In variousembodiments, the antisense oligomer conjugates are administered monthlyat 30 mg/kg. In various embodiments, the antisense oligomer conjugatesare administered monthly at 40 mg/kg. In some embodiments, the antisenseoligomer conjugates are administered monthly at 60 mg/kg. In someembodiments, the antisense oligomer conjugates are administered monthlyat 80 mg/kg. In some embodiments, the antisense oligomer conjugates areadministered monthly at 100 mg/kg. In some embodiments, the antisenseoligomer conjugates are administered monthly at 160 mg/kg. As usedherein, monthly is understood to have the art-accepted meaning of everymonth.

As would be understood in the art, weekly, biweekly, every third week,or monthly administrations may be in one or more administrations orsub-doses as discussed herein.

Nucleic acid molecules and antisense oligomer conjugates describedherein can be administered to cells by a variety of methods known tothose familiar to the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres, as described herein andknown in the art. In certain embodiments, microemulsification technologymay be utilized to improve bioavailability of lipophilic (waterinsoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo,S. K., et al., Drug Development and Industrial Pharmacy, 17(12),1685-1713, 1991) and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7),712-714, 1991). Among other benefits, microemulsification providesenhanced bioavailability by preferentially directing absorption to thelymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

In one aspect of disclosure, the formulations contain micelles formedfrom an oligomer as provided herein and at least one amphiphiliccarrier, in which the micelles have an average diameter of less thanabout 100 nm. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizean antisense oligomer conjugate of the present disclosure andmicroemulsify it at a later stage when the solution comes into a contactwith a complex water phase (such as one found in human gastro-intestinaltract). Usually, amphiphilic ingredients that satisfy these requirementshave HLB (hydrophilic to lipophilic balance) values of 2-20, and theirstructures contain straight chain aliphatic radicals in the range of C-6to C-20. Examples are polyethylene-glycolized fatty glycerides andpolyethylene glycols.

Examples of amphiphilic carriers include saturated and monounsaturatedpolyethyleneglycolyzed fatty acid glycerides, such as those obtainedfrom fully or partially hydrogenated various vegetable oils. Such oilsmay advantageously consist of tri-, di-, and mono-fatty acid glyceridesand di- and mono-poly(ethylene glycol) esters of the corresponding fattyacids, with a particularly preferred fatty acid composition includingcapric acid 4-10%, capric acid 3-9%, lauric acid 40-50%, myristic acid14-24%, palmitic acid 4-14%, and stearic acid 5-15%. Another usefulclass of amphiphilic carriers includes partially esterified sorbitanand/or sorbitol, with saturated or mono-unsaturated fatty acids(SPAN-series) or corresponding ethoxylated analogs (TWEEN-series).

Commercially available amphiphilic carriers may be particularly useful,including Gelucire-series, Labrafil, Labrasol, or Lauroglycol (allmanufactured and distributed by Gattefosse Corporation, Saint Priest,France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurate anddi-laurate, Lecithin, Polysorbate 80, etc. (produced and distributed bya number of companies in USA and worldwide).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the pharmaceutical compositions ofthe present disclosure into suitable host cells. In particular, thepharmaceutical compositions of the present disclosure may be formulatedfor delivery either encapsulated in a lipid particle, a liposome, avesicle, a nanosphere, a nanoparticle or the like. The formulation anduse of such delivery vehicles can be carried out using known andconventional techniques.

Hydrophilic polymers suitable for use in the present disclosure arethose which are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepoly(ethylene glycol) (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. In certain embodiments, polymershave a weight average molecular weight of from about 100 or 120 daltonsup to about 5,000 or 10,000 daltons, or from about 300 daltons to about5,000 daltons. In other embodiments, the polymer is poly(ethyleneglycol) having a weight average molecular weight of from about 100 toabout 5,000 daltons, or having a weight average molecular weight of fromabout 300 to about 5,000 daltons. In certain embodiments, the polymer isa poly(ethylene glycol) having a weight average molecular weight ofabout 750 daltons, for example PEG(750). Polymers may also be defined bythe number of monomers therein; a preferred embodiment of the presentdisclosure utilizes polymers of at least about three monomers, such PEGpolymers consisting of three monomers have a molecular weight ofapproximately 132 daltons.

Other hydrophilic polymers which may be suitable for use in the presentdisclosure include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present disclosurecomprises a biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7, or 8glucose units, designated by the Greek letter α, β, or γ, respectively.The glucose units are linked by α-1,4-glucosidic bonds. As a consequenceof the chair conformation of the sugar units, all secondary hydroxylgroups (at C-2, C-3) are located on one side of the ring, while all theprimary hydroxyl groups at C-6 are situated on the other side. As aresult, the external faces are hydrophilic, making the cyclodextrinswater-soluble. In contrast, the cavities of the cyclodextrins arehydrophobic, since they are lined by the hydrogen of atoms C-3 and C-5,and by ether-like oxygens. These matrices allow complexation with avariety of relatively hydrophobic compounds, including, for instance,steroid compounds such as 17α-estradiol (see, e.g., van Uden et al.Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). The complexation takesplace by Van der Waals interactions and by hydrogen bond formation. Fora general review of the chemistry of cyclodextrins, see, Wenz, Agnew.Chem. Int. Ed. Engl., 33:803-822 (1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μm.Oligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present disclosure relates to formulations comprisingliposomes containing an antisense oligomer conjugate of the presentdisclosure, where the liposome membrane is formulated to provide aliposome with increased carrying capacity. Alternatively or in addition,the antisense oligomer conjugate of the present disclosure may becontained within, or adsorbed onto, the liposome bilayer of theliposome. An antisense oligomer conjugate of the present disclosure maybe aggregated with a lipid surfactant and carried within the liposome'sinternal space; in these cases, the liposome membrane is formulated toresist the disruptive effects of the active agent-surfactant aggregate.

According to one embodiment of the present disclosure, the lipid bilayerof a liposome contains lipids derivatized with poly(ethylene glycol)(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present disclosure arein solubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent disclosure. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPGs) of varying chain lengths(for example, from about C14 to about C20). Polymer-derivatized lipidssuch as PEG-lipids may also be utilized for micelle formation as theywill act to inhibit micelle/membrane fusion, and as the addition of apolymer to surfactant molecules decreases the CMC of the surfactant andaids in micelle formation. Preferred are surfactants with CMOs in themicromolar range; higher CMC surfactants may be utilized to preparemicelles entrapped within liposomes of the present disclosure.

Liposomes according to the present disclosure may be prepared by any ofa variety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT application WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;and Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993. For example, liposomes of the presentdisclosure may be prepared by diffusing a lipid derivatized with ahydrophilic polymer into preformed liposomes, such as by exposingpreformed liposomes to micelles composed of lipid-grafted polymers, atlipid concentrations corresponding to the final mole percent ofderivatized lipid which is desired in the liposome. Liposomes containinga hydrophilic polymer can also be formed by homogenization, lipid-fieldhydration, or extrusion techniques, as are known in the art.

In another exemplary formulation procedure, the active agent is firstdispersed by sonication in a lysophosphatidylcholine or other low CMCsurfactant (including polymer grafted lipids) that readily solubilizeshydrophobic molecules. The resulting micellar suspension of active agentis then used to rehydrate a dried lipid sample that contains a suitablemole percent of polymer-grafted lipid, or cholesterol. The lipid andactive agent suspension is then formed into liposomes using extrusiontechniques as are known in the art, and the resulting liposomesseparated from the unencapsulated solution by standard columnseparation.

In one aspect of the present disclosure, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988). In certainembodiments, reagents such as DharmaFECT® and Lipofectamine® may beutilized to introduce polynucleotides or proteins into cells.

The release characteristics of a formulation of the present disclosuredepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In mostcases the amount should be between 0.1 and 30 percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range istypically between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

An antisense oligomer conjugate may be formulated to be containedwithin, or, adapted to release by a surgical or medical device orimplant. In certain aspects, an implant may be coated or otherwisetreated with an antisense oligomer conjugate. For example, hydrogels, orother polymers, such as biocompatible and/or biodegradable polymers, maybe used to coat an implant with the pharmaceutical compositions of thepresent disclosure (i.e., the composition may be adapted for use with amedical device by using a hydrogel or other polymer). Polymers andcopolymers for coating medical devices with an agent are well-known inthe art. Examples of implants include, but are not limited to, stents,drug-eluting stents, sutures, prosthesis, vascular catheters, dialysiscatheters, vascular grafts, prosthetic heart valves, cardiac pacemakers,implantable cardioverter defibrillators, IV needles, devices for bonesetting and formation, such as pins, screws, plates, and other devices,and artificial tissue matrices for wound healing.

In addition to the methods provided herein, the antisense oligomerconjugates for use according to the disclosure may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals. The antisense oligomerconjugates and their corresponding formulations may be administeredalone or in combination with other therapeutic strategies in thetreatment of muscular dystrophy, such as myoblast transplantation, stemcell therapies, administration of aminoglycoside antibiotics, proteasomeinhibitors, and up-regulation therapies (e.g., upregulation of utrophin,an autosomal paralogue of dystrophin).

In some embodiments, the additional therapeutic may be administeredprior, concurrently, or subsequently to the administration of theantisense oligomer conjugate of the present disclosure. For example, theantisense oligomer conjugates may be administered in combination with asteroid and/or antibiotic. In certain embodiments, the antisenseoligomer conjugates are administered to a patient that is on backgroundsteroid theory (e.g., intermittent or chronic/continuous backgroundsteroid therapy). For example, in some embodiments the patient has beentreated with a corticosteroid prior to administration of an antisenseoligomer and continues to receive the steroid therapy. In someembodiments, the steroid is glucocorticoid or prednisone.

The routes of administration described are intended only as a guidesince a skilled practitioner will be able to determine readily theoptimum route of administration and any dosage for any particular animaland condition. Multiple approaches for introducing functional newgenetic material into cells, both in vitro and in vivo have beenattempted (Friedmann (1989) Science, 244:1275-1280). These approachesinclude integration of the gene to be expressed into modifiedretroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research51(18), suppl.: 50745-5079S); integration into non-retrovirus vectors(e.g., adeno-associated viral vectors) (Rosenfeld, et al. (1992) Cell,68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or deliveryof a transgene linked to a heterologous promoter-enhancer element vialiposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med.Sci., 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288;Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; andWang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855);coupled to ligand-specific, cation-based transport systems (Wu and Wu(1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA,expression vectors (Nabel et al. (1990), supra; Wolff et al. (1990)Science, 247:1465-1468). Direct injection of transgenes into tissueproduces only localized expression (Rosenfeld (1992) supra; Rosenfeld etal. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; andHazinski et al. (1991) supra). The Brigham et al. group (Am. J. Med.Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) havereported in vivo transfection only of lungs of mice following eitherintravenous or intratracheal administration of a DNA liposome complex.An example of a review article of human gene therapy procedures is:Anderson, Science (1992) 256:808-813.

In a further embodiment, pharmaceutical compositions of the disclosuremay additionally comprise a carbohydrate as provided in Han et al., Nat.Comms. 7, 10981 (2016) the entirety of which is incorporated herein byreference. In some embodiments, pharmaceutical compositions of thedisclosure may comprise 5% of a hexose carbohydrate. For example,pharmaceutical composition of the disclosure may comprise 5% glucose, 5%fructose, or 5% mannose. In certain embodiments, pharmaceuticalcompositions of the disclosure may comprise 2.5% glucose and 2.5%fructose. In some embodiments, pharmaceutical compositions of thedisclosure may comprises a carbohydrate selected from: arabinose presentin an amount of 5% by volume, glucose present in an amount of 5% byvolume, sorbitol present in an amount of 5% by volume, galactose presentin an amount of 5% by volume, fructose present in an amount of 5% byvolume, xylitol present in an amount of 5% by volume, mannose present inan amount of 5% by volume, a combination of glucose and fructose eachpresent in an amount of 2.5% by volume, and a combination of glucosepresent in an amount of 5.7% by volume, fructose present in an amount of2.86% by volume, and xylitol present in an amount of 1.4% by volume.

IV. Methods of Use

Restoration of the Dystrophin Reading Frame Using Exon Skipping

A potential therapeutic approach to the treatment of DMD caused byout-of-frame mutations in the dystrophin gene is suggested by the milderform of dystrophinopathy known as BMD, which is caused by in-framemutations. The ability to convert an out-of-frame mutation to anin-frame mutation would hypothetically preserve the mRNA reading frameand produce an internally shortened yet functional dystrophin protein.Antisense oligomer conjugates of the disclosure were designed toaccomplish this.

Hybridization of the PMO with the targeted pre-mRNA sequence interfereswith formation of the pre-mRNA splicing complex and deletes exon 53 fromthe mature mRNA. The structure and conformation of antisense oligomerconjugates of the disclosure allow for sequence-specific base pairing tothe complementary sequence. By similar mechanism, eteplirsen, forexample, which is a PMO that was designed to skip exon 51 of dystrophinpre-mRNA allows for sequence-specific base pairing to the complementarysequence contained in exon 51 of dystrophin pre-mRNA.

Normal dystrophin mRNA containing all 79 exons will produce normaldystrophin protein. The graphic in FIG. 1 depicts a small section of thedystrophin pre-mRNA and mature mRNA, from exon 47 to exon 53. The shapeof each exon depicts how codons are split between exons; of note, onecodon consists of three nucleotides. Rectangular shaped exons start andend with complete codons. Arrow shaped exons start with a complete codonbut end with a split codon, containing only nucleotide #1 of the codon.Nucleotides #2 and #3 of this codon are contained in the subsequent exonwhich will start with a chevron shape.

Dystrophin mRNA missing whole exons from the dystrophin gene typicallyresult in DMD. The graphic in FIG. 2 illustrates a type of geneticmutation (deletion of exon 50) that is known to result in DMD. Sinceexon 49 ends in a complete codon and exon 51 begins with the secondnucleotide of a codon, the reading frame after exon 49 is shifted,resulting in out-of-frame mRNA reading frame and incorporation ofincorrect amino acids downstream from the mutation. The subsequentabsence of a functional C-terminal dystroglycan binding domain resultsin production of an unstable dystrophin protein.

Eteplirsen skips exon 51 to restore the mRNA reading frame. Since exon49 ends in a complete codon and exon 52 begins with the first nucleotideof a codon, deletion of exon 51 restores the reading frame, resulting inproduction of an internally-shortened dystrophin protein with an intactdystroglycan binding site, similar to an “in-frame” BMD mutation (FIG.3).

The feasibility of ameliorating the DMD phenotype using exon skipping torestore the dystrophin mRNA open reading frame is supported bynonclinical research. Numerous studies in dystrophic animal models ofDMD have shown that restoration of dystrophin by exon skipping leads toreliable improvements in muscle strength and function (Sharp 2011;Yokota 2009; Wu 2008; Wu 2011; Barton-Davis 1999; Goyenvalle 2004;Gregorevic 2006; Yue 2006; Welch 2007; Kawano 2008; Reay 2008; vanPutten 2012). A compelling example of this comes from a study in whichdystrophin levels following exon skipping (using a PMO) therapy werecompared with muscle function in the same tissue. In dystrophic mdxmice, tibialis anterior (TA) muscles treated with a mouse-specific PMOmaintained ˜75% of their maximum force capacity after stress-inducingcontractions, whereas untreated contralateral TA muscles maintained only˜25% of their maximum force capacity (p<0.05) (Sharp 2011). In anotherstudy, 3 dystrophic CXMD dogs received, at 2-5 months of age,exon-skipping therapy using a PMO-specific for their genetic mutationonce a week for 5 to 7 weeks or every other week for 22 weeks. Followingexon-skipping therapy, all 3 dogs demonstrated extensive, body-wideexpression of dystrophin in skeletal muscle, as well as maintained orimproved ambulation (15 m running test) relative to baseline. Incontrast, untreated age-matched CXMD dogs showed a marked decrease inambulation over the course of the study (Yokota 2009).

PMOs were shown to have more exon skipping activity at equimolarconcentrations than phosphorothioates in both mdx mice and in thehumanized DMD (hDMD) mouse model, which expresses the entire human DMDtranscript (Heemskirk 2009). In vitro experiments using reversetranscription polymerase chain reaction (RT-PCR) and Western blot (WB)in normal human skeletal muscle cells or muscle cells from DMD patientswith different mutations amenable to exon 51 skipping identifiedeteplirsen (a PMO) as a potent inducer of exon 51 skipping.Eteplirsen-induced exon 51 skipping has been confirmed in vivo in thehDMD mouse model (Arechavala-Gomeza 2007).

Clinical outcomes for analyzing the effect of an antisense oligomerconjugate that is complementary to a target region of exon 53 of thehuman dystrophin pre-mRNA and induces exon 53 skipping include percentdystrophin positive fibers (PDPF), six-minute walk test (6MWT), loss ofambulation (LOA), North Star Ambulatory Assessment (NSAA), pulmonaryfunction tests (PFT), ability to rise (from a supine position) withoutexternal support, de novo dystrophin production, and other functionalmeasures.

In some embodiments, the present disclosure provides methods forproducing dystrophin in a subject having a mutation of the dystrophingene that is amenable to exon 53 skipping, the method comprisingadministering to the subject an antisense oligomer conjugate, orpharmaceutically acceptable salt thereof, as described herein. Incertain embodiments, the present disclosure provides methods forrestoring an mRNA reading frame to induce dystrophin protein productionin a subject with Duchenne muscular dystrophy (DMD) who has a mutationof the dystrophin gene that is amenable to exon 53 skipping. Proteinproduction can be measured by reverse-transcription polymerase chainreaction (RT-PCR), western blot analysis, or immunohistochemistry (IHC).

In some embodiments, the present disclosure provides methods fortreating DMD in a subject in need thereof, wherein the subject has amutation of the dystrophin gene that is amenable to exon 53 skipping,the method comprising administering to the subject an antisense oligomerconjugate, or pharmaceutically acceptable salt thereof, as describedherein. In various embodiments, treatment of the subject is measured bydelay of disease progression. In some embodiments, treatment of thesubject is measured by maintenance of ambulation in the subject orreduction of loss of ambulation in the subject. In some embodiments,ambulation is measured using the 6 Minute Walk Test (6MWT). In certainembodiments, ambulation is measured using the North Start AmbulatoryAssessment (NSAA).

In various embodiments, the present disclosure provides methods formaintaining pulmonary function or reducing loss of pulmonary function ina subject with DMD, wherein the subject has a mutation of the DMD genethat is amenable to exon 53 skipping, the method comprisingadministering to the subject an antisense oligomer conjugate, orpharmaceutically acceptable salt thereof, as described herein. In someembodiments, pulmonary function is measured as Maximum ExpiratoryPressure (MEP). In certain embodiments, pulmonary function is measuredas Maximum Inspiratory Pressure (MIP). In some embodiments, pulmonaryfunction is measured as Forced Vital Capacity (FVC).

In a further embodiment, the pharmaceutical compositions of thedisclosure may be co-administered with a carbohydrate in the methods ofthe disclosure, either in the same formulation or is a separateformulation, as provided in Han et al., Nat. Comms. 7, 10981 (2016) theentirety of which is incorporated herein by reference. In someembodiments, pharmaceutical compositions of the disclosure may beco-administered with 5% of a hexose carbohydrate. For example,pharmaceutical compositions of the disclosure may be co-administeredwith 5% glucose, 5% fructose, or 5% mannose. In certain embodiments,pharmaceutical compositions of the disclosure may be co-administeredwith 2.5% glucose and 2.5% fructose. In some embodiments, pharmaceuticalcomposition of the disclosure may be co-administered with a carbohydrateselected from: arabinose present in an amount of 5% by volume, glucosepresent in an amount of 5% by volume, sorbitol present in an amount of5% by volume, galactose present in an amount of 5% by volume, fructosepresent in an amount of 5% by volume, xylitol present in an amount of 5%by volume, mannose present in an amount of 5% by volume, a combinationof glucose and fructose each present in an amount of 2.5% by volume, anda combination of glucose present in an amount of 5.7% by volume,fructose present in an amount of 2.86% by volume, and xylitol present inan amount of 1.4% by volume.

In various embodiments, an antisense oligomer conjugate of thedisclosure is co-administered with a therapeutically effective amount ofa non-steroidal anti-inflammatory compound. In some embodiments, thenon-steroidal anti-inflammatory compound is an NF-kB inhibitor. Forexample, in some embodiments, the NF-kB inhibitor may be CAT-1004 or apharmaceutically acceptable salt thereof. In various embodiments, theNF-kB inhibitor may be a conjugate of salicylate and DHA. In someembodiments, the NF-kB inhibitor is CAT-1041 or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the NF-kB inhibitor isa conjugate of salicylate and EPA. In various embodiments, the NF-kBinhibitor is

or a pharmaceutically acceptable salt thereof.

In some embodiments, non-steroidal anti-inflammatory compound is a TGF-binhibitor. For example, in certain embodiments, the TGF-b inhibitor isHT-100.

In certain embodiments, there is described an antisense oligomerconjugate as described herein for use in therapy. In certainembodiments, there is described an antisense oligomer conjugate asdescribed herein for use in the treatment of Duchenne musculardystrophy. In certain embodiments, there is described an antisenseoligomer conjugate as described herein for use in the manufacture of amedicament for use in therapy. In certain embodiments, there isdescribed an antisense oligomer conjugate as described herein for use inthe manufacture of a medicament for the treatment of Duchenne musculardystrophy.

V. Kits

The disclosure also provides kits for treatment of a patient with agenetic disease which kit comprises at least an antisense molecule(e.g., an antisense oligomer conjugate comprising the antisense oligomerset forth in SEQ ID NO: 1), packaged in a suitable container, togetherwith instructions for its use. The kits may also contain peripheralreagents such as buffers, stabilizers, etc. Those of ordinary skill inthe field should appreciate that applications of the above method haswide application for identifying antisense molecules suitable for use inthe treatment of many other diseases. In an or a embodiment, the kitcomprises an antisense oligomer conjugate according to Formula (III).

Examples

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this disclosure that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims. The following examples areprovided by way of illustration only and not by way of limitation. Thoseof skill in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results.

Materials and Methods

Cells and Tissue Culture Treatment Conditions

Differentiated human myocytes (ZenBio, Inc.) were utilized to measureexon skipping. Specifically, myoblasts (ZenBio, Inc., SKB-F) were grownto 80-90% confluence at 37° C. and 5% CO₂ in growth media (SKB-M;ZenBio, Inc.). Differentiation was initiated by replacing the growthmedia with differentiation media (SKM-D; ZenBio, Inc.). To assay exon 53skipping, 1×10⁴ differentiated cells were plated in a 24-well plate and1 mL of differentiation media (SKM-D; ZenBio, Inc.) containing variousconcentrations of PMO or PPMO was added to each well and incubated for96 hours.

Western Blot Analysis

For western blot analysis, tissue was homogenized with homogenizationbuffer (4% SDS, 4 M urea, 125 mM tris-HCl (pH 6.8)) at a ratio of 9 to18×20-μm tissue sections at approximately 5 mm in diameter in 133 μL ofbuffer. The corresponding lysate was collected and subjected to proteinquantification using the RC DC Protein Assay Kit per manufacturer'sinstructions (BioRad Cat. 500-0122). The tissue extract samples werediluted 1:10 using homogenization buffer to fall within the range of theBSA standard curve. Samples were prepared such that 35 μl of samplewould contain the desired amount of protein using 25 μl of proteinlysate, 7 μl NuPAGE LDS Sample Buffer (Life Technologies Cat. NP0008,Carlsbad, Calif., USA), and 3 μl NuPAGE Reducing Agent (10×) (LifeTechnologies Cat. NP0004). After heating the protein samples for 5minutes at 95° C., samples were centrifuged and supernatant was loadedonto a NuPAGE Novex 10 well, 1 mm, mini 3-8% polyacrylamide tris-acetategel (Life Technologies Cat. EA0375) at a maximum of 50 μg total proteinload per lane. The gel was run at 150 volts at room temperature untilthe dye front had run off the gel. The resulting protein gels weretransferred to PVDF membranes (Life Technologies Cat. LC2007) for 75minutes at room temperature with 30 volts using NuPAGE transfer buffer(Life Technologies NP006-1), 10% methanol and 0.1% NuPAGE antioxidant(Life Technologies NP0005).

After protein transfer, the PVDF membranes were immersed in TTBS buffer(1×TBS (Amresco Cat. J640-4L), 0.1% (v/v) tween-20). The membranes weretransferred to blocking buffer (5% (w/v) non-fat dry milk (LabScientific Cat. M0841) in TTBS) and soaked overnight at 4° C. withgentle rocking. After blocking, the membranes were incubated for either60 minutes at room temperature in DYS1 (Leica Cat. NCL-DYS1) diluted1:20 using blocking buffer, or 20 minutes at room temperature inanti-α-actinin antibody (Sigma-Aldrich Cat. NA931V) diluted 1:100,000with blocking buffer, followed by six washes (five minutes each withTTBS). Anti-mouse IgG conjugated to horseradish peroxidase (GEHealthcare Cat. NA931V) was diluted 1:40,000 using blocking buffer andadded to the membranes for 45 minutes (DYS1) or 15 minutes (α-actinin),followed again by six washes. Using the ECL Prime Western Detection Kit(GE Healthcare Cat. RPN2232), film was exposed to the gel and developedaccordingly. Developed film was scanned and analyzed using ImageQuant TLPlus software (version 8.1) and linear regression analysis was performedusing Graphpad software.

Each Western blot gel includes a 4 or 5 point dystrophin standard curveprepared using total protein extracted from normal tissue (mousequadriceps, diaphragm, or heart) diluted to, for example, 64%, 16%, 4%,1%, and 0.25% (see. For example. FIGS. 5A and 5B) and spiked into DMDtissue (for example, mdx mouse quadriceps, diaphragm, or heart, or NHPquadriceps, diaphragm, or smooth muscle (GI)) extract. Standard curvesamples were processed as described above. Dystrophin protein levels aspercent of wild-type dystrophin levels (% WT) were determined bycomparing dystrophin band intensities to the gel standard curve.

RT-PCR Analysis

For RT-PCR analysis, RNA was isolated from the cells using the IllustraGE spin kit following the manufacture's protocol. Concentration andpurity of the RNA was determined using a NanoDrop. Exon 53 skipping wasmeasured by RT-PCR with a forward primer that binds exon 51/52 junctionand 54 SEQ ID NO: 5 (5′-CATCAAGCAGAAGGCAACAA-3′) and a reverse primerthat binds exon 51/52 junction and 54 SEQ ID NO: 6(5′-GAAGTTTCAGGGCCAAGTCA-3′). A skipped exon 53 resulted in a 201 bpamplicon and an unskipped exon 53 resulted in a 413 bp amplicon.

Mouse exon 23 skipping was measured by RT-PCR with a forward primer-SEQID NO: 7 (5′-CACATCTTTGATGGTGTGAGG-3′) and a reverse primer SEQ ID NO: 8(5′-CAACTTCAGCCATCCATTTCTG-3′).

After the RNA was subjected to RT-PCR, the samples were analyzed using aCaliper machine, which uses gel capillary electrophoresis. Percent exonskipping was calculated using the following equation: (area under thecurve for skipped bands)/(sum of area under curve for skipped andunskipped bands)×100.

Immunohistochemistry: Dystrophin Staining:

10 micron frozen tissue sections of the mouse quadriceps were used todetect dystrophin by dystrophin primary antibody (dilution 1:250,rabbit, Abcam, cat #ab 15277) in 10% goat serum+1% BSA in PBS andsecondary antibody Alexa-Fluoro 488 goat anti-rabbit (dilution of1:1000) in 10% goat serum+1% BSA.

Preparation of Morpholino Subunits

Referring to Scheme 1, wherein B represents a base pairing moiety, themorpholino subunits may be prepared from the correspondingribinucleoside (1) as shown. The morpholino subunit (2) may beoptionally protected by reaction with a suitable protecting groupprecursor, for example trityl chloride. The 3′ protecting group isgenerally removed during solid-state oligomer synthesis as described inmore detail below. The base pairing moiety may be suitably protected forsolid-phase oligomer synthesis. Suitable protecting groups includebenzoyl for adenine and cytosine, phenylacetyl for guanine, andpivaloyloxymethyl for hypoxanthine (I). The pivaloyloxymethyl group canbe introduced onto the Ni position of the hypoxanthine heterocyclicbase. Although an unprotected hypoxanthine subunit, may be employed,yields in activation reactions are far superior when the base isprotected. Other suitable protecting groups include those disclosed inU.S. Pat. No. 8,076,476, which is hereby incorporated by reference inits entirety.

Reaction of 3 with the activated phosphorous compound 4 results inmorpholino subunits having the desired linkage moiety 5.

Compounds of structure 4 can be prepared using any number of methodsknown to those of skill in the art. Coupling with the morpholino moietythen proceeds as outlined above.

Compounds of structure 5 can be used in solid-phase oligomer synthesisfor preparation of oligomers comprising the intersubunit linkages. Suchmethods are well known in the art. Briefly, a compound of structure 5may be modified at the 5′ end to contain a linker to a solid support.Once supported, the protecting group of 5 (e.g., trityl at 3′-end)) isremoved and the free amine is reacted with an activated phosphorousmoiety of a second compound of structure 5. This sequence is repeateduntil the desired length oligo is obtained. The protecting group in theterminal 3′ end may either be removed or left on if a 3′ modification isdesired. The oligo can be removed from the solid support using anynumber of methods, or example treatment with a base to cleave thelinkage to the solid support.

The preparation of morpholino oligomers in general and specificmorpholino oligomers of the disclosure are described in more detail inthe Examples.

Preparation of Morpholino Oligomers

The preparation of the compounds of the disclosure are performed usingthe following protocol according to Scheme 2:

Preparation of trityl piperazine phenyl carbamate 35: To a cooledsuspension of compound 11 in dichloromethane (6 mL/g 11) was added asolution of potassium carbonate (3.2 eq) in water (4 mL/g potassiumcarbonate). To this two-phase mixture was slowly added a solution ofphenyl chloroformate (1.03 eq) in dichloromethane (2 g/g phenylchloroformate). The reaction mixture was warmed to 20° C. Upon reactioncompletion (1-2 hr), the layers were separated. The organic layer waswashed with water, and dried over anhydrous potassium carbonate. Theproduct 35 was isolated by crystallization from acetonitrile.

Preparation of carbamate alcohol 36: Sodium hydride (1.2 eq) wassuspended in 1-methyl-2-pyrrolidinone (32 mL/g sodium hydride). To thissuspension were added triethylene glycol (10.0 eq) and compound 35 (1.0eq). The resulting slurry was heated to 95° C. Upon reaction completion(1-2 hr), the mixture was cooled to 20° C. To this mixture was added 30%dichloromethane/methyl tert-butyl ether (v:v) and water. Theproduct-containing organic layer was washed successively with aqueousNaOH, aqueous succinic acid, and saturated aqueous sodium chloride. Theproduct 36 was isolated by crystallization from dichloromethane/methyltert-butyl ether/heptane.

Preparation of Tail acid 37: To a solution of compound 36 intetrahydrofuran (7 mL/g 36) was added succinic anhydride (2.0 eq) andDMAP (0.5 eq). The mixture was heated to 50° C. Upon reaction completion(5 hr), the mixture was cooled to 20° C. and adjusted to pH 8.5 withaqueous NaHCO₃. Methyl tert-butyl ether was added, and the product wasextracted into the aqueous layer. Dichloromethane was added, and themixture was adjusted to pH 3 with aqueous citric acid. Theproduct-containing organic layer was washed with a mixture of pH=3citrate buffer and saturated aqueous sodium chloride. Thisdichloromethane solution of 37 was used without isolation in thepreparation of compound 38.

Preparation of 38: To the solution of compound 37 was addedN-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq),4-dimethylaminopyridine (DMAP) (0.34 eq), and then1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1.1eq). The mixture was heated to 55° C. Upon reaction completion (4-5 hr),the mixture was cooled to 20° C. and washed successively with 1:1 0.2 Mcitric acid/brine and brine. The dichloromethane solution underwentsolvent exchange to acetone and then to N,N-dimethylformamide, and theproduct was isolated by precipitation from acetone/N,N-dimethylformamideinto saturated aqueous sodium chloride. The crude product was reslurriedseveral times in water to remove residual N,N-dimethylformamide andsalts.

PMO Synthesis Method A: Use of Disulfide Anchor

Introduction of the activated “Tail” onto the anchor-loaded resin wasperformed in dimethyl imidazolidinone (DMI) by the procedure used forincorporation of the subunits during solid phase synthesis.

This procedure was performed in a silanized, jacketed peptide vessel(ChemGlass, NJ, USA) with a coarse porosity (40-60 μm) glass frit,overhead stirrer, and 3-way Teflon stopcock to allow N2 to bubble upthrough the fit or a vacuum extraction.

The resin treatment/wash steps in the following procedure consist of twobasic operations: resin fluidization or stirrer bed reactor andsolvent/solution extraction. For resin fluidization, the stopcock waspositioned to allow N2 flow up through the frit and the specified resintreatment/wash was added to the reactor and allowed to permeate andcompletely wet the resin. Mixing was then started and the resin slurrymixed for the specified time. For solvent/solution extraction, mixingand N2 flow were stopped and the vacuum pump was started and then thestopcock was positioned to allow evacuation of resin treatment/wash towaste. All resin treatment/wash volumes were 15 mL/g of resin unlessnoted otherwise.

To aminomethylpolystyrene resin (100-200 mesh; ˜1.0 mmol/g load based onnitrogen substitution; 75 g, 1 eq, Polymer Labs, UK, part #1464-X799) ina silanized, jacketed peptide vessel was added 1-methyl-2-pyrrolidinone(NMP; 20 ml/g resin) and the resin was allowed to swell with mixing for1-2 hr. Following evacuation of the swell solvent, the resin was washedwith dichloromethane (2×1-2 min), 5% diisopropylethylamine in 25%isopropanol/dichloromethane (2×3-4 min) and dichloromethane (2×1-2 min).After evacuation of the final wash, the resin was treated with asolution of disulfide anchor 34 in 1-methyl-2-pyrrolidinone (0.17 M; 15mL/g resin, ˜2.5 eq) and the resin/reagent mixture was heated at 45° C.for 60 hr. On reaction completion, heating was discontinued and theanchor solution was evacuated and the resin washed with1-methyl-2-pyrrolidinone (4×3-4 min) and dichloromethane (6×1-2 min).The resin was treated with a solution of 10% (v/v) diethyl dicarbonatein dichloromethane (16 mL/g; 2×5-6 min) and then washed withdichloromethane (6×1-2 min). The resin 39 was dried under a N2 streamfor 1-3 hr and then under vacuum to constant weight (±2%). Yield:110-150% of the original resin weight.

Determination of the Loading of Aminomethylpolystyrene-disulfide resin:The loading of the resin (number of potentially available reactivesites) is determined by a spectrometric assay for the number oftriphenylmethyl (trityl) groups per gram of resin.

A known weight of dried resin (25±3 mg) is transferred to a silanized 25ml volumetric flask and ˜5 mL of 2% (v/v) trifluoroacetic acid indichloromethane is added. The contents are mixed by gentle swirling andthen allowed to stand for 30 min. The volume is brought up to 25 mL withadditional 2% (v/v) trifluoroacetic acid in dichloromethane and thecontents thoroughly mixed. Using a positive displacement pipette, analiquot of the trityl-containing solution (500 μL) is transferred to a10 mL volumetric flask and the volume brought up to 10 mL withmethanesulfonic acid.

The trityl cation content in the final solution is measured by UVabsorbance at 431.7 nm and the resin loading calculated in trityl groupsper gram resin (μmol/g) using the appropriate volumes, dilutions,extinction coefficient (c: 41 μmol-1 cm−1) and resin weight. The assayis performed in triplicate and an average loading calculated.

The resin loading procedure in this example will provide resin with aloading of approximately 500 μmol/g. A loading of 300-400 in μmol/g wasobtained if the disulfide anchor incorporation step is performed for 24hr at room temperature.

Tail loading: Using the same setup and volumes as for the preparation ofaminomethylpolystyrene-disulfide resin, the Tail can be introduced intosolid support. The anchor loaded resin was first deprotected underacidic condition and the resulting material neutralized before coupling.For the coupling step, a solution of 38 (0.2 M) in DMI containing4-ethylmorpholine (NEM, 0.4 M) was used instead of the disulfide anchorsolution. After 2 hr at 45° C., the resin 39 was washed twice with 5%diisopropylethylamine in 25% isopropanol/dichloromethane and once withDCM. To the resin was added a solution of benzoic anhydride (0.4 M) andNEM (0.4 M). After 25 min, the reactor jacket was cooled to roomtemperature, and the resin washed twice with 5% diisopropylethylamine in25% isopropanol/dichloromethane and eight times with DCM. The resin 40was filtered and dried under high vacuum. The loading for resin 40 isdefined to be the loading of the originalaminomethylpolystyrene-disulfide resin 39 used in the Tail loading.

Solid Phase Synthesis: Morpholino Oligomers were prepared on a GilsonAMS-422 Automated Peptide Synthesizer in 2 mL Gilson polypropylenereaction columns (Part #3980270). An aluminum block with channels forwater flow was placed around the columns as they sat on the synthesizer.The AMS-422 will alternatively add reagent/wash solutions, hold for aspecified time, and evacuate the columns using vacuum.

For oligomers in the range up to about 25 subunits in length,aminomethylpolystyrene-disulfide resin with loading near 500 μmol/g ofresin is preferred. For larger oligomers,aminomethylpolystyrene-disulfide resin with loading of 300-400 μmol/g ofresin is preferred. If a molecule with 5′-Tail is desired, resin thathas been loaded with Tail is chosen with the same loading guidelines.

The following reagent solutions were prepared:

Detritylation Solution: 10% Cyanoacetic Acid (w/v) in 4:1dichloromethane/acetonitrile;

Neutralization Solution: 5% Diisopropylethylamine in 3:1dichloromethane/isopropanol; and

Coupling Solution: 0.18 M (or 0.24 M for oligomers having grown longerthan 20 subunits) activated Morpholino Subunit of the desired base andlinkage type and 0.4 M N ethylmorpholine, in1,3-dimethylimidazolidinone.

Dichloromethane (DCM) was used as a transitional wash separating thedifferent reagent solution washes.

On the synthesizer, with the block set to 42° C., to each columncontaining 30 mg of aminomethylpolystyrene-disulfide resin (or Tailresin) was added 2 mL of 1-methyl-2-pyrrolidinone and allowed to sit atroom temperature for 30 min. After washing with 2 times 2 mL ofdichloromethane, the following synthesis cycle was employed:

Step Volume Delivery Hold time Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mL Manifold15 seconds Detritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mLManifold 15 seconds Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds Neutralization 1.5 mL Manifold 30 seconds Neutralization 1.5mL Manifold 30 seconds Neutralization 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsCoupling 350-500 uL Syringe 40 minutes DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds DCM 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsDCM 1.5 mL Manifold 30 seconds

The sequences of the individual oligomers were programmed into thesynthesizer so that each column receives the proper coupling solution(A,C,G,T,I) in the proper sequence. When the oligomer in a column hadcompleted incorporation of its final subunit, the column was removedfrom the block and a final cycle performed manually with a couplingsolution comprised of 4-methoxytriphenylmethyl chloride (0.32 M in DMI)containing 0.89 M 4-ethylmorpholine.

Cleavage from the resin and removal of bases and backbone protectinggroups: After methoxytritylation, the resin was washed 8 times with 2 mL1-methyl-2-pyrrolidinone. One mL of a cleavage solution consisting of0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in1-methyl-2-pyrrolidinone was added, the column capped, and allowed tosit at room temperature for 30 min. After that time, the solution wasdrained into a 12 mL Wheaton vial. The greatly shrunken resin was washedtwice with 300 μL of cleavage solution. To the solution was added 4.0 mLconc. aqueous ammonia (stored at −20° C.), the vial capped tightly (withTeflon lined screw cap), and the mixture swirled to mix the solution.The vial was placed in a 45° C. oven for 16-24 hr to effect cleavage ofbase and backbone protecting groups.

Crude product purification: The vialed ammonolysis solution was removedfrom the oven and allowed to cool to room temperature. The solution wasdiluted with 20 mL of 0.28% aqueous ammonia and passed through a 2.5×10cm column containing Macroprep HQ resin (BioRad). A salt gradient (A:0.28% ammonia with B: 1 M sodium chloride in 0.28% ammonia; 0-100% B in60 min) was used to elute the methoxytrityl containing peak. Thecombined fractions were pooled and further processed depending on thedesired product.

Demethoxytritylation of Morpholino Oligomers: The pooled fractions fromthe Macroprep purification were treated with 1 M H₃PO₄ to lower the pHto 2.5. After initial mixing, the samples sat at room temperature for 4min, at which time they are neutralized to pH 10-11 with 2.8%ammonia/water. The products were purified by solid phase extraction(SPE).

SPE column packing and conditioning: Amberchrome CG-300M (Rohm and Haas;Philadelphia, Pa.) (3 mL) is packed into 20 mL fritted columns (BioRadEcono-Pac Chromatography Columns (732-1011)) and the resin rinsed with 3mL of the following: 0.28% NH₄OH/80% acetonitrile; 0.5 M NaOH/20%ethanol; water; 50 mM H₃PO₄/80% acetonitrile; water; 0.5 NaOH/20%ethanol; water; 0.28% NH₄OH.

SPE purification: The solution from the demethoxytritylation was loadedonto the column and the resin rinsed three times with 3-6 mL 0.28%aqueous ammonia. A Wheaton vial (12 mL) was placed under the column andthe product eluted by two washes with 2 mL of 45% acetonitrile in 0.28%aqueous ammonia.

Product isolation: The solutions were frozen in dry ice and the vialsplaced in a freeze dryer to produce a fluffy white powder. The sampleswere dissolved in water, filtered through a 0.22 micron filter (PallLife Sciences, Acrodisc 25 mm syringe filter, with a 0.2 micron HTTuffryn membrane) using a syringe and the Optical Density (OD) wasmeasured on a UV spectrophotometer to determine the OD units of oligomerpresent, as well as dispense sample for analysis. The solutions werethen placed back in Wheaton vials for lyophilization.

Analysis of Morpholino Oligomers by MALDI: MALDI-TOF mass spectrometrywas used to determine the composition of fractions in purifications aswell as provide evidence for identity (molecular weight) of theoligomers. Samples were run following dilution with solution of3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid),3,4,5-trihydoxyacetophenone (THAP) or alpha-cyano-4-hydoxycinnamic acid(HCCA) as matrices.

PMO Synthesis Method B: Use of NCP2 Anchor

NCP2 Anchor Synthesis: 1. Preparation of Methyl 4-Fluoro-3-Nitrobenzoate(1)

To a 100 L flask was charged 12.7 kg of 4-fluoro-3-nitrobenzoic acid wasadded 40 kg of methanol and 2.82 kg concentrated sulfuric acid. Themixture was stirred at reflux (65° C.) for 36 hours. The reactionmixture was cooled to 0° C. Crystals formed at 38° C. The mixture washeld at 0° C. for 4 hrs then filtered under nitrogen. The 100 L flaskwas washed and filter cake was washed with 10 kg of methanol that hadbeen cooled to 0° C. The solid filter cake was dried on the funnel for 1hour, transferred to trays, and dried in a vacuum oven at roomtemperature to a constant weight of 13.695 kg methyl4-fluoro-3-nitrobenzoate (100% yield; HPLC 99%).

2. Preparation of 3-Nitro-4-(2-oxopropyl)benzoic Acid A. (Z)-Methyl4-(3-Hydroxy-1-Methoxy-1-Oxobut-2-en-2-yl)-3-Nitrobenzoate (2)

To a 100 L flask was charged 3.98 kg of methyl 4-fluoro-3-nitrobenzoate(1) from the previous step 9.8 kg DMF, 2.81 kg methyl acetoacetate. Themixture was stirred and cooled to 0° C. To this was added 3.66 kg DBUover about 4 hours while the temperature was maintained at or below 5°C. The mixture was stirred an additional 1 hour. To the reaction flaskwas added a solution of 8.15 kg of citric acid in 37.5 kg of purifiedwater while the reaction temperature was maintained at or below 15° C.After the addition, the reaction mixture was stirred an addition 30minutes then filtered under nitrogen. The wet filter cake was returnedto the 100 L flask along with 14.8 kg of purified water. The slurry wasstirred for 10 minutes then filtered. The wet cake was again returned tothe 100 L flask, slurried with 14.8 kg of purified water for 10 minutes,and filtered to crude (Z)-methyl4-(3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl)-3-nitrobenzoate.

B. 3-Nitro-4-(2-oxopropyl)benzoic Acid

The crude (Z)-methyl4-(3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl)-3-nitrobenzoate was chargedto a 100 L reaction flask under nitrogen. To this was added 14.2 kg1,4-dioxane and the stirred. To the mixture was added a solution of16.655 kg concentrated HCl and 13.33 kg purified water (6 M HCl) over 2hours while the temperature of the reaction mixture was maintained below15° C. When the addition was complete, the reaction mixture was heatedat reflux (80° C.) for 24 hours, cooled to room temperature, andfiltered under nitrogen. The solid filter cake was triturated with 14.8kg of purified water, filtered, triturated again with 14.8 kg ofpurified water, and filtered. The solid was returned to the 100 L flaskwith 39.9 kg of DCM and refluxed with stirring for 1 hour. 1.5 kg ofpurified water was added to dissolve the remaining solids. The bottomorganic layer was split to a pre-warmed 72 L flask, then returned to aclean dry 100 L flask. The solution was cooled to 0° C., held for 1hour, then filtered. The solid filter cake was washed twice each with asolution of 9.8 kg DCM and 5 kg heptane, then dried on the funnel. Thesolid was transferred to trays and dried to a constant weight of 1.855kg 3-Nitro-4-(2-oxopropyl)benzoic Acid. Overall yield 42% fromcompound 1. HPLC 99.45%.

3. Preparation of N-Tritylpiperazine Succinate (NTP)

To a 72 L jacketed flask was charged under nitrogen 1.805 kgtriphenylmethyl chloride and 8.3 kg of toluene (TPC solution). Themixture was stirred until the solids dissolved. To a 100 L jacketedreaction flask was added under nitrogen 5.61 kg piperazine, 19.9 kgtoluene, and 3.72 kg methanol. The mixture was stirred and cooled to 0°C. To this was slowly added in portions the TPC solution over 4 hourswhile the reaction temperature was maintained at or below 10° C. Themixture was stirred for 1.5 hours at 10° C., then allowed to warm to 14°C. 32.6 kg of purified water was charged to the 72 L flask, thentransferred to the 100 L flask while the internal batch temperature wasmaintained at 20+/−5° C. The layers were allowed to split and the bottomaqueous layer was separated and stored. The organic layer was extractedthree times with 32 kg of purified water each, and the aqueous layerswere separated and combined with the stored aqueous solution.

The remaining organic layer was cooled to 18° C. and a solution of 847 gof succinic acid in 10.87 kg of purified water was added slowly inportions to the organic layer. The mixture was stirred for 1.75 hours at20+/−5° C. The mixture was filtered, and the solids were washed with 2kg TBME and 2 kg of acetone then dried on the funnel. The filter cakewas triturated twice with 5.7 kg each of acetone and filtered and washedwith 1 kg of acetone between triturations. The solid was dried on thefunnel, then transferred to trays and dried in a vacuum oven at roomtemperature to a constant weight of 2.32 kg of NTP. Yield 80%.

4. Preparation of(4-(2-Hydroxypropyl)-3-Nitrophenyl)(4-Tritylpiperazin-1-yl)MethanonePreparation of1-(2-Nitro-4(4-Tritylpiperazine-1-Carbony)Phenyl)Propan-2-one

To a 100 L jacketed flask was charged under nitrogen 2 kg of3-Nitro-4-(2-oxopropyl)benzoic Acid (3), 18.3 kg DCM, and 1.845 kgN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl).The solution was stirred until a homogenous mixture was formed. 3.048 kgof NTP was added over 30 minutes at room temperature and stirred for 8hours. 5.44 kg of purified water was added to the reaction mixture andstirred for 30 minutes. The layers were allowed to separate and thebottom organic layer containing the product was drained and stored. Theaqueous layer was extracted twice with 5.65 kg of DCM. The combinedorganic layers were washed with a solution of 1.08 kg sodium chloride in4.08 kg purified water. The organic layers were dried over 1.068 kg ofsodium sulfate and filtered. The sodium sulfate was washed with 1.3 kgof DCM. The combined organic layers were slurried with 252 g of silicagel and filtered through a filter funnel containing a bed of 252 g ofsilica gel. The silica gel bed was washed with 2 kg of DCM. The combinedorganic layers were evaporated on a rotovap. 4.8 kg of THE was added tothe residue and then evaporated on the rotovap until 2.5 volumes of thecrude 1-(2-nitro-4(4-tritylpiperazine-1-carbonyl)phenyl)propan-2-one inTHF was reached.

B. Preparation of(4-(2-Hydroxypropyl)-3-Nitrophenyl)(4-Tritylpiperazin-1-yl)Methanone (5)

To a 100 L jacketed flask was charged under nitrogen 3600 g of 4 fromthe previous step and 9800 g THF. The stirred solution was cooled to <5°C. The solution was diluted with 11525 g ethanol and 194 g of sodiumborohydride was added over about 2 hours at <5° C. The reaction mixturewas stirred an additional 2 hours at <5° C. The reaction was quenchedwith a solution of about 1.1 kg ammonium chloride in about 3 kg of waterby slow addition to maintain the temperature at <10° C. The reactionmixture was stirred an additional 30 minutes, filtered to removeinorganics, and recharged to a 100 L jacketed flask and extracted with23 kg of DCM. The organic layer was separated and the aqueous was twicemore extracted with 4.7 kg of DCM each. The combined organic layers werewashed with a solution of about 800 g of sodium chloride in about 3 kgof water, then dried over 2.7 kg of sodium sulfate. The suspension wasfiltered and the filter cake was washed with 2 kg of DCM. The combinedfiltrates were concentrated to 2.0 volumes, diluted with about 360 g ofethyl acetate, and evaporated. The crude product was loaded onto asilica gel column of 4 kg of silica packed with DCM under nitrogen andeluted with 2.3 kg ethyl acetate in 7.2 kg of DCM. The combinedfractions were evaporated and the residue was taken up in 11.7 kg oftoluene. The toluene solution was filtered and the filter cake waswashed twice with 2 kg of toluene each. The filter cake was dried to aconstant weight of 2.275 kg of compound 5 (46% yield from compound 3)HPLC 96.99%.

5. Preparation of2,5-Dioxopyrrolidin-1-yl(1-(2-Nitro-4-(4-triphenylmethylpiperazine-1Carbonyl)Phenyl)Propan-2-yl) Carbonate (NCP2 Anchor)

To a 100 L jacketed flask was charged under nitrogen 4.3 kg of compound5 (weight adjusted based on residual toluene by H¹ NMR; all reagentshere after were scaled accordingly) and 12.7 kg pyridine. To this wascharged 3.160 kg of DSC (78.91 weight % by H¹ NMR) while the internaltemperature was maintained at <35° C. The reaction mixture was aged forabout 22 hours at ambience then filtered. The filter cake was washedwith 200 g of pyridine. In two batches each comprising ½ the filtratevolume, filtrate wash charged slowly to a 100 L jacketed flaskcontaining a solution of about 11 kg of citric acid in about 50 kg ofwater and stirred for 30 minutes to allow for solid precipitation. Thesolid was collected with a filter funnel, washed twice with 4.3 kg ofwater per wash, and dried on the filter funnel under vacuum.

The combined solids were charged to a 100 L jacketed flask and dissolvedin 28 kg of DCM and washed with a solution of 900 g of potassiumcarbonate in 4.3 kg of water. After 1 hour, the layers were allowed toseparate and the aqueous layer was removed. The organic layer was washedwith 10 kg of water, separated, and dried over 3.5 kg of sodium sulfate.The DCM was filtered, evaporated, and dried under vacuum to 6.16 kg ofNCP2 Anchor (114% yield).

NCP2 Anchor Loaded Resin Synthesis

To a 75 L solid phase synthesis reactor with a Teflon stop cock wascharged about 52 L of NMP and 2300 g of aminomethyl polystyrene resin.The resin was stirred in the NMP to swell for about 2 hours thendrained. The resin was washed twice with about 4 L DCM per wash, thentwice with 39 L Neutralization Solution per wash, then twice with 39 Lof DCM per wash. The NCP2 Anchor Solution was slowly added to thestirring resin solution, stirred for 24 hours at room temperature, anddrained. The resin was washed four times with 39 L of NMP per wash, andsix times with 39 L of DCM per wash. The resin was treated and stirredwith ½ the DEDC Capping Solution for 30 minutes, drained, and wastreated and stirred with the 2^(nd) ½ of the DEDC Capping Solution for30 minutes and drained. The resin was washed six times with 39 L of DCMper wash then dried in an oven to constant weight of 3573.71 g of AnchorLoaded Resin. Preparation of Morpholino Oligomer using NCP2 Anchor

50 L Solid-phase Synthesis of Golodirsen (PMO #1) Crude Drug Substance

1 Materials

TABLE 2 Starting Materials Material Chemical CAS Chemical Molecular NameName Number Formula Weight Activated Phosphoramidochloridic acid,1155373-30-0 C₃₈H₃₇ClN₇O₄P 722.2 A N, N-dimethyl-, [6-[6- Subunit(benzoylamino)-9H-purin-9-yl]- 4-(triphenylmethyl)-2- morpholinyl]methylester Activated Phosphoramidochloridic acid, 1155373-31-1 C₃₇H₃₇ClN₅O₅P698.2 C Subunit N, N-dimethyl-, [6-[4- (benzoylamino)-2-oxo-1(2H)-pyrimidinyl]-4- (triphenylmethyl)-2- morpholinyl]methyl ester ActivatedPropanoic Acid, 2, 2-dimethyl-, 1155309-89-9 C₅₁H₅₃ClN₇O₇P 942.2 DPG4-[[[9-[6- Subunit [[[chloro(dimethylamino)phosp hinyl]oxy]methyl]-4-(triphenylmethyl)-2- morpholinyl]-2-[(2- phenylacetyl)amino]-9H-purin-6-yl]oxy]methyl]phenyl ester Activated Phosphoramidochloridic acid,1155373-34-4 C₃₁H₃₄ClN₄O₅P 609.1 T Subunit N, N-dimethyl-, 6-(3,4-dihydro- 5-methyl-2, 4-dioxo-1(2H)- pyrimidinyl)]-4-(triphenylmethyl)-2- morpholinyl]methyl ester Activated Butanedioicacid, 1- 1380600-06-5 C₄₃H₄₇N₃O₁₀ 765.9 EG3 Tail [3aR, 45, 7R, 7a5)-1,3, 3a, 4,7, 7a- hexahydro-1, 3-dioxo-4, 7- methano-2H-isoindol-2-yl]-4-[2-[2-[2-[[[4-(triphenylmethyl)- 1-piperazinyl]carbonyl]oxy]ethoxy]ethoxy]ethyl] ester

Chemical Structures of Starting Materials:

A. Activated EG3 Tail

B. Activated C Subunit (for Preparation, See U.S. Pat. No. 8,067,571)

C. Activated a Subunit (for Preparation, See U.S. Pat. No. 8,067,571)

D. Activated DPG Subunit (for Preparation, See WO 2009/064471)

E. Activated T Subunit (for Preparation, See WO 2013/082551)

F Anchor Loaded Resin

wherein R^(l) is a support-medium.

TABLE 3 Description of Solutions for Solid Phase Oligomer Synthesis ofGolodirsen Crude Drug Substance Solution Name Solution Composition NCP2Anchor 37.5 L NMP and 1292 g NCP2 Anchor Solution DEDC Capping 4.16 LDiethyl Dicarbonate (DEDC), 3.64 L NEM, and 33.8 L DCM Solution CYTFASolution 2.02 kg 4-cyanopyridine, 158 L DCM, 1.42 L TFA, 39 L TFE, and 2L purified water Neutralization 35.3 L IPA, 7.5 L DIPEA, and 106.5 L DCMSolution Cleavage Solution 1,530.04 g DTT, 6.96 L NMP, and 2.98 L DBU

2. Synthesis of Golodirsen Crude Drug Substance

A. Resin Swelling

750 g of Anchor Loaded Resin and 10.5 L of NMP were charged to a 50 Lsilanized reactor and stirred for 3 hours. The NMP was drained and theAnchor Loaded Resin was washed twice with 5.5 L each of DCM and twicewith 5.5 L each of 30% TFE/DCM.

B. Cycle 0: EG3 Tail Coupling

The Anchor Loaded Resin was washed three times with 5.5 L each of 30%TFE/DCM and drained, washed with 5.5 L of CYFTA solution for 15 minutesand drained, and again washed with 5.5 L of CYTFA Solution for 15minutes without draining to which 122 mL of 1:1 NEM/DCM was charged andthe suspension stirred for 2 minutes and drained. The resin was washedtwice with 5.5 L of Neutralization Solution for 5 minutes and drained,then twice with 5.5 L each of DCM and drained. A solution of 706.2 g ofactivated EG3 Tail (MW 765.85) and 234 mL of NEM in 3 L of DMI wascharged to the resin and stirred for 3 hours at RT and drained. Theresin was washed twice with 5.5 L each of Neutralization Solution for 5minutes per each wash, and once with 5.5 L of DCM and drained. Asolution of 374.8 g of benzoic anhydride and 195 mL NEM in 2680 mL NMPwas charged and stirred for 15 minutes and drained. The resin wasstirred with 5.5 L of Neutralization Solution for 5 minutes, then washedonce with 5.5 L of DCM and twice with 5.5 L each of 30% TFE/DCM. Theresin was suspended in 5.5 L of 30% TFE/DCM and held for 14 hours.

C. Subunit Coupling Cycles 1-30

i. Pre-Coupling Treatments

Prior to each coupling cycle as described in FIG. 18, the resin was: 1)washed with 30% TFE/DCM; 2) a) treated with CYTFA Solution 15 minutesand drained, and b) treated with CYTFA solution for 15 minutes to whichwas added 1:1 NEM/DCM, stirred, and drained; 3) stirred three times withNeutralization Solution; and 4) washed twice with DCM. See FIG. 18.

ii. Post Coupling Treatments

After each subunit solution was drained as described in FIG. 18, theresin was: 1) washed with DCM; and 2) washed two times with 30% TFE/DCM.If the resin was held for a time period prior to the next couplingcycle, the second TFE/DCM wash was not drained and the resin wasretained in said TFE/DCM wash solution. See FIG. 18.

iii. Activated Subunit Coupling Cycles

The coupling cycles were performed as described in FIG. 18.

iv. Final IPA Washing

After the final coupling step was performed as described in FIG. 18, theresin was washed 8 times with 19.5 L each of IPA, and dried under vacuumat room temperature for about 63.5 hours to a dried weight of 4857.9 g.

C. Cleavage

The above resin bound Golodirsen Crude Drug Substance was divided intotwo lots, each lot was treated as follows. A 1619.3 g lot of resinwas: 1) stirred with 10 L of NMP for 2 hrs, then the NMP was drained; 2)washed tree times with 10 L each of 30% TFE/DCM; 3) treated with 10 LCYTFA Solution for 15 minutes; and 4) 10 L of CYTFA Solution for 15minutes to which 130 ml 1:1 NEM/DCM was then added and stirred for 2minutes and drained. The resin was treated three times with 10 L each ofNeutralization Solution, washed six times with 10 L of DCM, and eighttimes with 10 L each of NMP. The resin was treated with a CleavingSolution of 1530.4 g DTT and 2980 DBU in 6.96 L NMP for 2 hours todetach the Eteplisen Crude Drug Substance from the resin. The Cleavingsolution was drained and retained in a separate vessel. The reactor andresin were washed with 4.97 L of NMP which was combined with theCleaving Solution.

D. Deprotection

The combined Cleaving Solution and NMP wash were transferred to apressure vessel to which was added 39.8 L of NH₄OH (NH₃.H₂O) that hadbeen chilled to a temperature of −10° C. to −25° C. in a freezer. Thepressure vessel was sealed and heated to 45° C. for 16 hrs then allowedto cool to 25° C. This deprotection solution containing the Golodirsencrude drug substance was diluted 3:1 with purified water and pH adjustedto 3.0 with 2 M phosphoric acid, then to pH 8.03 with NH₄OH. HPLC: C1877.552% and SCX-10 73.768%.

Purification of Golodirsen (PMO #1) Crude Drug Substance

The deprotection solution from above part D, containing the Golodirsencrude drug substance was loaded onto a column of ToyoPearl Super-Q 650Sanion exchange resin (Tosoh Bioscience) and eluted with a gradient of0-35% B over 17 column volume (Buffer A: 10 mM sodium hydroxide; BufferB: 1 M sodium chloride in 10 mM sodium hydroxide) and fractions ofacceptable purity (C18 and SCX HPLC) were pooled to a purified drugproduct solution. HPLC: 93.571% (C18) 88.270% (SCX).

The purified drug substance solution was desalted and lyophilized to1450.72 g purified Golodirsen drug substance. Yield 54.56%; HPLC:93.531% (C₁₈) 88.354% (SCX).

TABLE 5 Acronyms Acronym Name DBU 1, 8-Diazabicycloundec-7-ene DCMDichloromethane DIPEA N, N-Diisopropylethylamine DMI 1,3-Dimethyl-2-imidazolidinone DTT Dithiothreitol IPA Isopropyl alcohol MWMolecular weight NEM N-Ethylmorpholine NMP N-Methyl-2-pyrrolidone RTRoom temperature TFA 2, 2, 2-Trifluoroacetic acid TFE 2, 2,2-Trifluoroethanol

Representative Example of CPP Conjugation

Analytical Procedures: Matrix-assisted laser desorption ionizationtime-of-flight mass spectra (MALDI-TOF-MS) were recorded on a BrukerAutoflex™ Speed, using a sinapinic acid (SA) matrix. SCX-HPLC wasperformed on a Thermo Dionex UltiMate 3000 system equipped with a 3000diode array detector and a ProPac™ SCX-20 column (250×4 mm) using a flowrate of 1.0 mL/min (pH=2; 30° C. column temperature). The mobile phaseswere A (25% acetonitrile in water containing 24 mM H₃PO₄) and B (25%acetonitrile in water containing 1 M KCl and 24 mM H₃PO₄). Gradientelution was employed: 0 min, 35% B; 2 min, 35% B; 22 min, 80% B; 25 min,80% B; 25.1 min, 35% B; 30 min, 35% B.

To a mixture of the PMO4658 (1.82 g, 0.177 mmol, freshly dried bylyophilization for two days; SEQ ID NO: 11),Ac-L-Arg-L-Arg-L-Arg-L-Arg-L-Arg-L-Arg-Gly-OH hexatrifluoroacetate(614.7 mg, 0.354 mmol), and1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU, 134.4 mg, 0.354 mmol) was addeddimethyl sulfoxide (DMSO, 20 mL). The mixture was stirred at roomtemperature for 3 minutes, then N,N-diisopropylethylamine (DIPEA, 68.5mg, 0.530 mmol) was added. After 5 minutes, the cloudy mixture became aclear solution. The reaction was monitored by SCX-HPLC. After 2 hours,20 mL of 10% ammonium hydroxide solution (2.8% NH₃) was added. Themixture was stirred at room temperature for an additional 2 hours. Thereaction was terminated by the addition of 400 mL water.Trifluoroethanol (2.0 mL) was added to the solution.

The solution was divided into two portions and each portion was purifiedby a WCX column (10 g resin per column). Each WCX column was firstwashed with 20% acetonitrile in water (v/v) to remove the PMO4658starting material. The washings (225 mL for each column) were stoppedwhen MALDI-TOF mass spectrum analysis showed the absence of PMO4658signal. Each column was then washed with water (100 mL per column). Thedesired product, PPMO4658, was eluted by 2.0 M guanidine HCl (140 mL foreach column). The purified solutions of PPMO4658 were pooled togetherand then divided into two portions and each desalted by an SPE column(10 g resin for each column).

The SPE column was first washed with 1.0 M aqueous NaCl solution (100 mLfor each column) to generate the hexahydrochloride salt of PPMO4658.Each SPE column was then washed with water (200 mL for each column). Thefinal desalted PPMO4658 was eluted by 50% acetonitrile in water (v/v,150 mL for each column). The acetonitrile was removed by evacuation atreduced pressure. The resulting aqueous solution was lyophilized toobtain the desired conjugate PPMO4658 hexahydrochloride (1.93 g, 94.5%yield).

Example 1: PMO #1

Using the PMO synthesis method B protocol described above, PMO #1 wassynthesized:

where each Nu from 1 to 25 and 5′ to 3′ is:

Position No. 5’ to 3’ Nu 1 G 2 T 3 T 4 G 5 C 6 C 7 T 8 C 9 C 10 G 11 G12 T 13 T 14 C 15 T 16 G 17 A 18 A 19 G 20 G 21 T 22 G 23 T 24 T 25 Cwherein A is

C is

G is

and T is

HPLC: 71.85%; Conditions: Dionex DNAPac (DNX #97) Gradient: 75% A+20%B+5% C at 0 min; 50% A at 20 min; 25% A+75% C at 21 min; Mobile phase A:10 mM NaOH/20 mM NaCl; C: 10 mM NaOH/0.5 M NaCL. Column Temp: 45 C;Flowrate 1.0 mL/min.

Example 2: PPMO #1

Using the protocol described above for preparation of PPMO4658, PPMO #1was synthesized from PMO #1:

where each Nu from 1 to 25 and 5′ to 3′ is:

Position No. 5’ to 3’ Nu 1 G 2 T 3 T 4 G 5 C 6 C 7 T 8 C 9 C 10 G 11 G12 T 13 T 14 C 15 T 16 G 17 A 18 A 19 G 20 G 21 T 22 G 23 T 24 T 25 Cwherein A is

C is

G is

and T is

Example 3: Exon 53 Skipping In Vitro (Myoblasts)

Two compounds that target human dystrophin (DMD) exon 53 as described inthe table below, PMO #1 and PPMO #1 both of which contain the samesequence, were assessed for DMD exon 53 skipping in healthy humanmyoblasts.

Sequences of PMO#1 and PPMO#1  for human DMD exon 53. Targeting TS  SEQID Name  Sequence (TS)  NO. 5′ 3′ PMO#1 GTTGCCTCCGGTTCTGAAG 1 EG3 HGTGTTC  PPMO#1 GTTGCCTCCGGTTCTGAAG 1 EG3 -G-R6 GTGTTC 

Specifically, healthy human myoblasts (passage 5-6, SKB-F-SL purchasedfrom Zen-Bio, Inc.) were plated at ˜40% confluency when treated with PMO#1 or PPMO #1 at various concentrations (i.e., 40 μm, 20 μm, 10 μm, 5μm, 2.5 μm, and 1.25 μm) in SKM-M media (Zen-Bio, Inc.). Afterninety-six hours of incubation, myoblasts were washed with PBS and lysedby RA1 lysis buffer in the Illustra GE RNAspin 96 kit (Cat #25-055-75,GE Healthcare Bio-Sciences). Total RNA were isolated per manufacturer'srecommendation, except that 404, RNase-free water was used to elute RNA.

To determine exon 53 skipping by both compounds, two-step end-pointRT-PCR was performed. Specifically, eleven microliters of total RNA wasfirst reverse transcribed to cDNA by SuperScript IV First-strandsynthesis kit (Cat #18091200, Invitrogen) using random hexamers as perthe manufacturer's instructions. PCR was performed by adding 9 μL cDNAinto Platinum Taq DNA polymerase PCR Supermix High Fidelity (Cat#12532024, Invitrogen) with primers that targeted human DMD exons 51/52junction and 54 (forward primer: (SEQ ID NO: 5): CATCAAGCAGAAGGCAACAA;reverse primer: (SEQ ID NO: 6): GAAGTTTCAGGGCCAAGTCA). PCR amplificationwas performed using BioRad CFX96 real time thermocycler using theprogram shown in Table 2. Expression of the skipped or non-skipped PCRproducts were assessed by loading 324, PCR product onto LabChip GXsystem using DNA High Sensitivity Reagent kit (CLS760672, Perkin Elmer).Percentage of DMD exon 53 skipping is calculated as the percentage ofthe molarity (nmol/l) for exon 53 skipped band (201 bp) compared to thesum molarity for the skipped (201 bp) and the unskipped (413 bp) bands.

Two-tailed, unpaired Student's t-test (homoscedastic) was used to assesswhether the means of the 2 groups are statistically different from eachother at each dose. P-value <0.05 is considered as statisticallysignificant.

Thermocycler program used to amplify DMD amplicons with or without exon53 skipping. Step Temperature Time 1. Denature 94° C.  2 min 2. Denature94° C. 30 sec  3. Anneal 61.2° C.   30 sec  4. Extend 68° C.  1 min 5.Repeat step 2-4 34 cycles 6. Final Extension 68° C.  5 min 7. Store  4°C. ∞

The results are presented in the table below and in FIG. 4. In FIG. 4,error bars presents mean±SD, “[Number] x” above the bars denotesrelative fold-change in percentage of exon skipping by PPMO #1 comparedto PMO #1 at each concentration, and “*” indicates significantdifference between PMO #1 and PPMO #1 with p-value <0.05.

Percentage of DMD exon 53 skipping by PMO #1 and PPMO #1 in humanmyoblasts. Compound/ Percent Exon Skipping (mean ± SD) Dose (μm) 1.252.5 5 10 20 40 PMO #1 1.27 ± 0.24 2.19 ± 0.44 3.58 ± 0.91 6.56 ± 1.3211.06 ± 2.08 19.57 ± 4.23 PPMO #1 2.03 ± 0.48 3.73 ± 0.70 6.57 ± 1.4710.27 ± 2.78  17.08 ± 4.61 26.90 ± 4.78

The data in the table above and in FIG. 4 surprisingly show thatsignificantly higher exon 53 skipping results in myoblasts when thecells are treated with PPMO #1 as compared to PMO #1 at allconcentrations, the degree of which is unexpected. This significantimprovement is further demonstrated in the in vivo comparative non-humanprimate (NHP) study of Example 5 where NHPs were treated with PPMO #1 orPMO #1 and exon 53 skipping was measured in various relevant muscletissues (see Example 5 for details). Moreover, because PPMO #1decomposes in the SKM-M media used in this example (data not shown) onthe time scale of this study, the NHP study in fact demonstrates an evengreater improvement than the one demonstrated in this example.

Example 4: MDX Mouse Study

The mdx mouse is an accepted and well-characterized animal model forDuchene muscular dystrophy (DMD) containing a mutation in exon 23 of thedystrophin gene. The M23D antisense sequence (SEQ ID NO: 2) is known toinduce exon 23 skipping and restore of functional dystrophin expression.MDX mice at 6-7 weeks of age were given a single injection into the tailvein of either a PPMO4225 or PMO4225 of the table below at a dose of 40mg/kg, or with saline.

TS  SEQ ID Name Targeting Sequence (TS) NO. 5′ 3′ PMO4225GGCCAAACCTCGGCTTACCTGAAAT 2 EG3 H PPMO4225 GGCCAAACCTCGGCTTACCTGAAAT 2EG3 -G-R6PMO4225 and PPMO4225 were each prepared by PMO Method A and CPPconjugation methods described above.

Treated mice were sacrificed at 7, 30, 60 and 90 days post single doseinjection (n=6 per group). The diaphragm, heart and right quadricepswere processed for western blot analysis to measure production ofdystrophin protein and RT-PCR analysis to measure percentage of exonskipping, and the left quadriceps was processed for immunohistochemistryand H/E staining as described above.

Dystrophin protein restoration was quantified by western blot, andpercentage of exon 23 skipping was measured by RT-PCR each as describedabove.

RT-PCR and Western Blot results are shown in FIGS. 5A-10B and in thetables below. Surprisingly, PPMO4225 induced significantly higher andsustained levels of dystrophin restoration and exon 23 skipping comparedto PMO4225, with highest levels occurring at 30-days post injection.Even more surprising, PPMO4225 increased dystrophin levels in the heartwhen PMO4225 did not; dystrophin and exon skipping were not observed inthe heart at all time points with PMO4225.

  Quantification of Dystrophin Protein as Percentage of Wild TypeProtein (% WT) by Western Blot Compound PMO4225 PPMO4225 Day 7 30 60 907 30 60 90 Tissue Quadriceps 1.1 2.3 1.6 0.7 20.7 28.1 20.8 8.2Diaphragm 1.4 1.9 1.3 0.6 14.5 15.2 9.8 2.3 Heart 0 0 0 0 2.0 1.0 0.90.1

  Percent Exon Skipping as Measured by RT-PCR Compound PMO4225 PPMO4225Day 7 30 60 90 7 30 60 90 Tissue Quadriceps 21.2 5.5 7.9 2.8 61.5 42.0228.8 6.9 Diaphragm 29.9 2.6 0.5 0 51.6 36.76 3.05 0 Heart 0 0 0 0 13.152.64 0 0

Immunohistochemistry results are shown in FIG. 11. Here, PPMO4225restores dystrophin throughout the quadriceps, whereas 4225 produces a‘patchy-like’ pattern of expression. The uniform distribution ofdystrophin with PPMO4225 treatment indicates that widespread targetingof skeletal muscle is achievable. PPMO4225 has significantly improveddelivery over PMO4225 in vivo.

Example 5: Exon 53 Skipping in NHP

To further demonstrate the efficacy of exon skipping of PPMO antisenseoligomers, non-human primates are utilized. Specifically, cynomolgusmonkeys having intact muscle tissues were injected intravenously, withPPMO #1, PMO #1 (from Example 3), or saline according to the dosingschedule in the below table:

Cynomolgus Dosing Schedule Number Dose per Group Compound (mg/kg) groupDelivery Strategy 1 Saline  0 2

2 PPMO #1 20 3 Once weekly dosing, 4 total doses 3 PPMO #1 40 3 Animalsare sacrificed 4 PPMO #1 80 3 48 hours after last 5 PMO #1 40 3 dose, onday 24

Animals were observed throughout the study, including clinicalobservations (e.g., evaluation of skin and fur, respiratory effects) andbody weight measurements. Blood and urine samples were taken at leastbefore testing begins, and 24 hours after the first dose and last dose(where applicable).

At each scheduled necropsy, or euthanized in extremis, sections ofdiaphragm, smooth muscle of the duodenum, colon, and and aorta,quadriceps, deltoid, bicep, and heart were collected and snap frozen.Percent exon 53 skipping was determined using RT-PCR as described abovewith primers that targeted human DMD exons 51-54 (forward primer: TGCCAT CTC CAA ACT AGA AAT GCC A (SEQ ID NO. 9); reverse primer: GCG GAGGTC TTT GGC CAA CT (SEQ ID NO: 10)). Results are shown in FIGS. 20-27,and in the table below.

Percent Exon Skipping PPMO #1 PMO #1 Muscle 20 mg/kg 40 mg/kg 80 mg/kg40 mg/kg Quadriceps 35.59 72.94 95.37  2.64 Diaphragm 64.64 96.06 99.17 2.23 Biceps 36.7  93.26 96.91  2.34 Deltoid 48.01 88.89 98.1   2.54Heart 2917 71.53 92.57  1.32 Aorta 53.81 73.77 73.53 30.47 Duodenum15.75 46.94 74.99  6.94 Colon 27.81 67.98 87.81  8.42

Surprisingly, PPMO #1 produced profound levels of exon skipping in theintact tissues tested as compared to PMO #1. Specifically, robust exon53 skipping was observed in skeletal, cardiac and smooth muscles withPPMO #1. Weekly dosing of PMO #1 at 40 mg/kg produced only low levels ofexon 53 skipping in skeletal muscle and heart (1.3-2.6%), and moderatelevels in smooth muscle (6.9-30.5%).

Notably and unexpectedly, whereas PMO #1 administration result inskipping between 1.32 to 2.64% in the quadriceps, diaphragm, biceps,deltoid and heart, PPMO #1 produced significantly and substantiallyhigher exon skipping, for example, in excess of at least 10× higher at20 mg/kg, at least 27× higher at 40 mg/kg, and in excess of 90% exon 53skipping in all these tissues at the 80 mg/kg dosage level. Particularlysurprising is the level of exon skipping achieved in the heart at, forexample, 80 mg/kg where exon skipping was in excess of 90% whereas PMO#1 produced 1.32% at 40 mg/kg. Without wishing to be bound by anyparticular theory, systematic administration and delivery of PPMO #1into the intact non-dystrophic NHP muscle tissues and achievement ofexon 53 skipping to the degree achieved by PPMO #1 particularly incardiac muscle could not have been predicted from the above mdx mouse inExample 4. Rather, deliver to healthy tissue as in the NHP differs fromdelivery to dystrophic tissue.

Example 6: MDX Mouse Dose Response Study

MDX mice at 6-7 weeks of age were given a single injection into the tailvein of either a PPMO4225 or PMO4225 described above at a dose of 40mg/kg, 80 mg/kg, or 120 mg/kg (n=6 per group).

Treated mice were sacrificed at 30 days post injection. The diaphragm,quadriceps, and heart were processed for western blot analysis tomeasure production of dystrophin protein based on the above-describedwestern blot protocol (used, for example, in Example 4) with thefollowing modifications:

Western Blot Western Blot Protocol of Protocol Parameter Example 4modifications Protein quantification RC DC Protein Assay Kit BCA methodBlocking Step Overnight at 4° C 1 Hour at RT Primary Antibody Incubation1 hour at RT Overnight at 4° C. Primary Antibody 1:20 1:500Concentration

Dystrophin protein restoration as % wild type is presented in the tablebelow and in FIGS. 12-15.

  Quantification of Dystrophin Protein as Percentage of Wild TypeProtein (% WT) by Western Blot Compound PMO4225 PPMO4225 Dose (mg/kg) 4080 120 40 80 120 Tissue Diaphragm 0.80 0.97 1.83 8.02 26.03 42.77 Heart0.13 0.24 0.34 0.61 6.34 19.48 Quadriceps 3.5 2.6 3.0 43 90 144

Surprisingly, the data shows that a single dose of PPMO4225 increasesdystrophin levels in a dose-dependent manner in mdx mice tosignificantly and substantially greater extent than PMO4225.

Example 7: MDX Mouse IHC Study of Diaphragm and Heart

MDX mice at 6-7 weeks of age were given a single injection into the tailvein of PPMO4225 at a dose of 80 mg/kg or saline, and wild type mice at6-7 weeks of age were given a single injection of saline. The treatedmdx mice, saline mdx mice, and wild type mice were sacrificed at 30 dayspost single dose injection (n=4 per group). Immunohistochemistry resultsare shown in FIG. 19. Here, the results show uniform increase indystrophin in tissues associated with morbidity and mortality in DMD inmdx mice treated with PPMO4225.

Example 8: Exon 53 Skipping In Vitro (Myotubes)

Two compounds that target human dystrophin (DMD) exon 53 as described inthe table below, PMO #1 and PPMO #1 both of which contain the samesequence were assessed for DMD exon 53 skipping in healthy humanmyotubes.

Sequences of PMO#1 and PPMO#1  for human DMD exon 53. Targeting sequence Name Compound Sequence  ID NO. 5′ 3′ PMO#1GTT GCC TCC GGT TCT GAA H53 EG3 H GGT GTT C  (+36+60) PPMO#1GTT GCC TCC GGT TCT GAA  H53 EG3 GR GGT GTT C  (+36+60  6Ac R6G)

Specifically, healthy human myoblasts (passage 5-6, SKB-F-SL purchasedfrom Zen-Bio, Inc.) were cultured to reach 80-90% confluency in SKM-Mmedia prior to initiation of differentiation by incubating in low serummedia (SKM-D, Zen-Bio, Inc.) Five-days after differentiation, maturemyotubes were incubated with the above compounds at variousconcentrations (i.e., 40 μm, 20 μm, 10 μm, 5 μm, 2.5 μm, and 1.25 μm).After ninety-six hours of incubation, myotubes were washed with PBS andlysed by RLT lysis buffer from the RNeasy Micro kit (Cat #74004,Qiagen). Total RNA were isolated per manufacturer's recommendation,except that 204, RNase-free water was used to elute RNA.

To determine exon 53 skipping by both compounds, two-step end-pointRT-PCR was performed. Specifically, seven microliters of total RNA wasfirst reverse transcribed to cDNA by SuperScript IV First-strandsynthesis kit (Cat #18091200, Invitrogen) using random hexamers as perthe manufacturer's instructions. PCR was performed by adding 5 μL cDNAinto Platinum Taq DNA polymerase PCR Supermix High Fidelity (Cat#12532024, Invitrogen) with primers that targeted human DMD exons 51/52junction and 54 (forward primer: CAT CAA GCA GAA GGC AAC AA; reverseprimer: GAA GTT TCA GGG CCA AGT CA). PCR amplification was performedusing BioRad CFX96 real time thermocycler using the program shown inTable 2. Expression of the skipped or non-skipped PCR products wereassessed by loading 324, PCR product onto LabChip GX system using DNAHigh Sensitivity Reagent kit (CLS760672, Perkin Elmer). Percentage ofDMD exon 53 skipping is calculated as the percentage of the molarity(nmol/l) for exon 53 skipped band (201 bp) compared to the sum molarityfor the skipped (201 bp) and the unskipped (413 bp) bands.

Two-tailed, unpaired Student's t-test (homoscedastic) was used to assesswhether the means of the 2 groups are statistically different from eachother at each dose. P-value <0.05 is considered as statisticallysignificant.

Thermocycler program used to amplify DMD amplicons with or without exon53 skipping. Step Temperature Time 8. Denature 94° C.  2 min 9. Denature94° C. 30 sec  10. Anneal 61.2° C.   30 sec  11. Extend 68° C.  1 min12. Repeat step 2-4 34 cycles 13. Final Extension 68° C.  5 min 14.Store  4° C. ∞

The results are presented in the table below and in FIG. 20. In FIG. 20,error bars presents mean±SD, “[Number] x” denotes relative fold-changein percentage of exon skipping by PPMO #1 compared to PMO #1 at eachconcentration, and “*,” “**” indicate significant difference between PMO#1 and PPMO #1 with p-value <0.05 or 0.005, respectively.

Percentage of DMD exon 53 skipping by PMO #1 and PPMO #1 in humanmyotubes. Compound/ Percent Exon Skipping (mean ± SD) Dose (μm) 1.25 2.55 10 20 40 PMO #1 1.45 ± 0.39 2.63 ± 0.50 3.33 ± 0.47 4.78 ± 0.88 8.40 ±0.53 12.98 ± 1.21 PPMO #1 3.10 ± 1.67 2.45 ± 0.52 4.73 ± 1.01 7.08 ±0.48 9.45 ± 1.03 12.08 ± 1.30

The data in the table above and in FIG. 24 surprisingly show thatsignificantly higher exon 53 skipping results in myotubes when the cellsare treated with PPMO #1 as compared to PMO #1 at least at 5 μm and 10μm concentrations, the degree of which is unexpected. This significantimprovement is further demonstrated in the in vivo comparative non-humanprimate (NHP) study of Example 5 where NHPs were treated with PPMO #1 orPMO #1 and exon 53 skipping was measured in various relevant muscletissues (see Example 5 for details). Moreover, because PPMO #1decomposes in the SKM-M media used in this example (data not shown) onthe time scale of this study, the NHP study in fact demonstrates an evengreater improvement than the one demonstrated in this example.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

REFERENCES{1,2}

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SEQUENCE LISTING SEQ Sequence 5′ to 3′ or ID DescriptionN terminus to C terminus NO H53A(+36+60) GTTGCCTCCGGTTCTGAAGGTGTTC 1mdx4225 GGCCAAACCTCGGCTTACCTGAAAT 2 R6 RRRRRR 3 R6-G RRRRRRG 4Human exon  CATCAAGCAGAAGGCAACAA 5 51/52 junction  and 54 bindingforward primer Human exon  GAAGTTTCAGGGCCAAGTCA 6 51/52 junction  and 54binding forward primer Mouse exon  CACATCTTTGATGGTGTGAGG 7 23 binding forward primer Mouse exon CAACTTCAGCCATCCATTTCTG 8 23 binding reverseprimer Human exon  TGCCATCTCCAAACTAGAAATGCCA 9 51-54 junction  bindingforward  Primer Human exon  GCGGAGGTCTTTGGCCAACT 10 51-54 junction binding reverse primer 4658 CTCCAACATCAAGGAAGATGGCATTTCTAG 11

1. An antisense oligomer conjugate of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: each Nu is anucleobase which taken together form a targeting sequence; and T is amoiety selected from:

R¹ is C₁-C₆ alkyl; wherein the targeting sequence is complementary to anexon 53 annealing site in the dystrophin pre-mRNA designated asH53A(+36+60).
 2. The antisense oligomer conjugate of claim 1, whereineach Nu is independently selected from cytosine (C), guanine (G),thymine (T), adenine (A), 5-methylcytosine (5 mC), uracil (U), andhypoxanthine (I).
 3. The antisense oligomer conjugate of claim 1,wherein the targeting sequence is SEQ ID NO: 1(5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′), wherein each thymine (T) isoptionally uracil (U).
 4. The antisense oligomer conjugate of claim 1,wherein T is

and the targeting sequence is SEQ ID NO: 1(5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′), wherein each thymine (T) isoptionally uracil (U).
 5. The antisense oligomer conjugate of claim 1,wherein T is

and the targeting sequence is SEQ ID NO: 1(5′-GTTGCCTCCGGTTCTGAAGGTGTTC-3′).
 6. An antisense oligomer conjugate ofFormula (II):

or a pharmaceutically acceptable salt thereof, wherein each Nu from 1 to25 and 5′ to 3′ is: Position No. 5’ to 3’ Nu 1 G 2 X 3 X 4 G 5 C 6 C 7 X8 C 9 C 10 G 11 G 12 X 13 X 14 C 15 X 16 G 17 A 18 A 19 G 20 G 21 X 22 G23 X 24 X 25 C

and wherein A is

C is

G is

and each X is independently


7. The antisense oligomer conjugate of claim 6, wherein each X is


8. The antisense oligomer conjugate of claim 6, wherein the antisenseoligomer is of Formula (IIA):

wherein each Nu from 1 to 25 and 5′ to 3′ is: Position No. 5’ to 3’ Nu 1G 2 X 3 X 4 G 5 C 6 C 7 X 8 C 9 C 10 G 11 G 12 X 13 X 14 C 15 X 16 G 17A 18 A 19 G 20 G 21 X 22 G 23 X 24 X 25 C

and wherein A is

C is

G is

and each X is independently


9. The antisense oligomer conjugate of claim 8, wherein each X is


10. An antisense oligomer conjugate of Formula (IV):

or a pharmaceutically acceptable salt thereof.
 11. The antisenseoligomer conjugate of claim 9, wherein the antisense oligomer is ofFormula (IVA):


12. A pharmaceutical composition, comprising an antisense oligomerconjugate of claim 1, or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier.
 13. A method for treatingDuchenne muscular dystrophy (DMD) in a subject in need thereof whereinthe subject has a mutation of the dystrophin gene that is amenable toexon 53 skipping, the method comprising administering to the subject theantisense oligomer conjugate of claim
 1. 14. The method of claim 13,wherein the antisense oligomer conjugate is administered weekly.
 15. Themethod of claim 13, wherein the antisense oligomer conjugate isadministered biweekly.
 16. The method of claim 13, wherein the antisenseoligomer conjugate is administered every third week.
 17. The method ofclaim 13, wherein the antisense oligomer conjugate is administeredmonthly.
 18. A method of restoring an mRNA reading frame to inducedystrophin production in a subject having a mutation of the dystrophingene that is amenable to exon 53 skipping, the method comprisingadministering to the subject the antisense oligomer conjugate ofclaim
 1. 19. The method of claim 18, wherein the antisense oligomerconjugate is administered weekly.
 20. The method of claim 18, whereinthe antisense oligomer conjugate is administered biweekly.
 21. Themethod of claim 18, wherein the antisense oligomer conjugate isadministered every third week.
 22. The method of claim 18, wherein theantisense oligomer conjugate is administered monthly.
 23. The method ofclaim 18, wherein the antisense oligomer conjugate is administered at adose of about 30 mg/kg.
 24. The method of claim 18, wherein theantisense oligomer conjugate is administered at a dose of about 40mg/kg.
 25. The method of claim 18, wherein the antisense oligomerconjugate is administered at a dose of about 60 mg/kg.
 26. The method ofclaim 18, wherein the antisense oligomer conjugate is administered at adose of about 80 mg/kg.
 27. The method of claim 18, wherein theantisense oligomer conjugate is administered at a dose of about 160mg/kg.
 28. A method for treating Duchenne muscular dystrophy (DMD) in asubject in need thereof wherein the subject has a mutation of thedystrophin gene that is amenable to exon 53 skipping, the methodcomprising administering to the subject the pharmaceutical compositionof claim
 12. 29. A method of restoring an mRNA reading frame to inducedystrophin production in a subject having a mutation of the dystrophingene that is amenable to exon 53 skipping, the method comprisingadministering to the subject the pharmaceutical composition of claim 12.30. A method of excluding exon 53 from dystrophin pre-mRNA during mRNAprocessing in a subject having a mutation of the dystrophin gene that isamenable to exon 53 skipping, the method comprising administering to thesubject the pharmaceutical composition of claim
 12. 31. A method ofbinding exon 53 of dystrophin pre-mRNA in a subject having a mutation ofthe dystrophin gene that is amenable to exon 53 skipping, the methodcomprising administering to the subject the pharmaceutical compositionof claim 12.